In this section, we systematically document the equipment, reagent sources, experimental procedures and reagent formulations required for our project. We carried out a series of workflows ranging from DNA manipulation (plasmid extraction, transformation, PCR, etc.) to protein expression, purification, and detection, supplemented by multiple analytical methods (electrophoresis, Western Blot, enzyme activity assays, etc.) to validate our results.
To ensure reproducibility and reliability, we provide detailed operation guidelines for instruments, list the sources of key reagents and kits, and include standard formulations for culture media and buffers. Together, these elements establish a complete, transparent, and reproducible experimental framework.
Protocol
DNA Lab
Plasmid Extraction
Plasmid MiniPrep Kit
Overnight bacterial culture
Double-distilled water
1.5 mL and 2 mL Eppendorf tubes
Equipment:Bench-top microcentrifuge
NanoDrop spectrophotometer
Procedure:1. Harvest the overnight bacterial culture (~5 mL) in an Eppendorf tube and centrifuge at 8000 × g for 2 min. Discard the supernatant.
2. Resuspend the bacterial pellet in 250 μL Buffer RB (colorless, containing RNase A). Transfer the suspension into two 2 mL Eppendorf tubes.
3. Add 250 μL Buffer LB (blue) to each tube and gently invert several times to lyse the cells.
4. Add 350 μL Buffer NB (yellow) and invert gently until a tight yellow precipitate forms. Incubate at room temperature for ~2 min.
5. Centrifuge at 12,000 × g for 5 min. Carefully transfer the supernatant to a spin column.
6. Centrifuge at 12,000 × g for 1 min. Discard the flow-through and repeat once.
7. Add 650 μL Buffer WB to the spin column, centrifuge at 12,000 × g for 1 min, and repeat once.
8. Centrifuge at 12,000 × g for 2 min to remove residual wash buffer. Leave the column open at room temperature for 5 min to dry.
9. Place the spin column in a new 1.5 mL tube (with cap removed). Add 30 μL preheated (70 °C) Buffer EB directly to the center of the membrane. Incubate at room temperature for 1 min.
10. Centrifuge at 12,000 × g for 1 min to elute the plasmid DNA.
11. Measure plasmid concentration and purity using a NanoDrop spectrophotometer (use EB as blank). Record dsDNA content.
Notes:- Ensure all centrifugation steps are carried out at room temperature.
- For optimal elution, Buffer EB should be prewarmed.
- Avoid disturbing the pellet or precipitate during transfer steps to ensure plasmid purity.
Plasmid Heat Shock Transformation
- Competent cells
- 1.5 mL Eppendorf tubes
- Plasmid DNA
- LB liquid medium
- LB agar plates
- Water bath or dry bath incubator
- Shaker incubator
- Incubator
1. Transfer ~50 μL of competent cells into a 1.5 mL Eppendorf tube.
2. Once thawed, add no more than 42.5 ng plasmid DNA to the cells and mix gently.
3. Incubate the mixture on ice for 30 min. For BL21(DE3) cells, place the tube in a 42 °C water bath (or dry bath) for 45 s, then immediately transfer back to ice for 2 min.
4. Add 800 μL LB liquid medium to the tube, seal with Parafilm, and mix gently.
5. Incubate the culture at 37 °C, ~200 rpm, for 1 h to allow recovery.
6. Take 50 μL of the culture, dilute with ddH₂O to a final volume of 100 μL, and spread 50 μL onto an LB agar plate.
7. Incubate the plates overnight at 37 °C in an inverted position.
Notes:- Ensure competent cells remain on ice until heat shock to maintain transformation efficiency.
- Do not exceed the recommended plasmid DNA amount, as high concentrations may reduce efficiency.
- Plates should be completely dry before spreading to avoid colony smearing.
Polymerase Chain Reaction (PCR)
- PCR tubes
- DNA template
- Forward and reverse primers
- ddH₂O
- High-fidelity DNA polymerase mix
- PCR thermocycler
1. Prepare the PCR reaction mix in a PCR tube as follows (depending on desired reaction volume):
• Template DNA: 1 μL
• Forward primer: 0.2 / 0.4 / 1 μL
• Reverse primer: 0.2 / 0.4 / 1 μL
• PCR Mix: 5 / 10 / 25 μL
• ddH₂O: add to a final volume of 10 / 20 / 50 μL
(Choose reaction volume based on experimental requirements).
2. Place the PCR tube(s) into the thermocycler.
3. Set up the PCR program:
• Denaturation: ~94–95 °C
• Annealing: set according to primer Tm value
• Extension: adjust based on fragment size (typically 1–2 kb/min at 72 °C)
• Number of cycles: 35
4. Start the run and ensure the program operates normally.
5. After completion, remove the PCR tube(s) for downstream applications.
Notes:- Use nuclease-free reagents to prevent contamination.
- Always keep PCR reagents on ice before starting to minimize nonspecific amplification.
- Optimize annealing temperature and Mg²⁺ concentration if nonspecific bands occur.
Colony PCR
- PCR tubes
- Bacterial suspension or single colony
- Forward and reverse primers
- ddH₂O
- High-fidelity DNA polymerase mix
- PCR thermocycler
1. Template preparation
• For bacterial suspension: Take 1 µL of suspension and dilute it in 9 µL ddH₂O.
• For single colony: Pick a single colony (obtained from streak plate or dilution plating) and resuspend in 10 µL ddH₂O.
2. Reaction setup
• Add the following components into a PCR tube (depending on desired reaction volume):
- Diluted bacterial suspension: 1 μL
- Forward primer: 0.2 / 0.4 / 1 μL
- Reverse primer: 0.2 / 0.4 / 1 μL
- PCR Mix: 5 / 10 / 25 μL
- ddH₂O: to a final volume of 10 / 20 / 50 μL
3. Thermocycler loading
• Place the PCR tube into the thermocycler.
4. Program setup
• Initial denaturation: ~94–95 °C
• Annealing: set according to primer Tm value
• Extension: 1–2 kb/min at 72 °C, depending on amplicon size
• Number of cycles: 35
5. Run and completion
• Start the program and monitor normal operation;
• After completion, remove PCR tubes for subsequent analysis.
- Ensure colonies are well-isolated to avoid cross-contamination.
- Use fresh colonies (12–24 h) for optimal DNA template quality.
- If PCR fails, perform a quick cell lysis (e.g., boiling for 5 min) before use as template.
Agarose Gel Electrophoresis
- Agarose powder
- 1× TAE buffer
- GelStain (nucleic acid dye, 10,000× stock)
- Loading buffer
- Gel casting tray
- Comb
- DNA marker/ladder
- Electrophoresis apparatus
- BG-gdsUVIEW Ultraviolet analyzer (or equivalent UV transilluminator)
1. Gel preparation
• To prepare a 1.0% agarose gel (100 mL total volume), weigh 1.0 g agarose powder into an Erlenmeyer flask.
• Add 100 mL of 1× TAE buffer.
• Heat the mixture in a microwave until boiling, swirling intermittently until the agarose is completely dissolved.
• Add the appropriate volume of 10,000× GelStain, mix thoroughly.
• Allow the solution to cool to ~60 °C (warm to touch but not hot).
2. Casting the gel
• Clean and dry the gel casting tray.
• Pour the agarose solution into the tray and insert the comb at the designated slot.
• Let the gel solidify completely.
• After polymerization, carefully remove the comb vertically.
• Transfer the gel (with tray) into the electrophoresis tank and cover with 1× TAE buffer.
3. Sample loading
• Mix DNA samples with appropriate volume of loading buffer.
• Using a micropipette, carefully load the samples into the wells.
• Replace pipette tips between samples to avoid cross-contamination.
• Load DNA marker/ladder into the first well.
4. Electrophoresis
• Connect the gel tank to the power supply and run at 90 V.
• Stop electrophoresis when the bromophenol blue dye front has migrated to ~1 cm from the bottom of the gel (typically ~20 minutes).
5. Visualization and analysis
• Remove the gel and place it onto the UV transilluminator.
• Observe DNA bands under UV illumination.
• Compare migration distances with the DNA ladder to determine fragment sizes.
- Ensure uniform gel casting to avoid uneven migration.
- Do not overload wells to prevent smearing.
- Always wear UV-protective goggles when observing gels under UV light.
Infusion Cloning
- 1.5 mL microcentrifuge tubes
- DNA fragment(s)
- Linearized vector
- 2× In-Fusion enzyme mix
- ddH₂O
- Benchtop microcentrifuge
- Dry bath incubator
1. Reaction setup
• In a 1.5 mL microcentrifuge tube, assemble the reaction mixture according to the following proportions:
- 10 μL of 2× In-Fusion enzyme mix
- 50–100 ng of linearized vector
- Insert DNA fragment at a molar ratio of 2–3× relative to the vector
- Add ddH₂O to a final volume of 20 μL
2. Mixing
• Briefly centrifuge the reaction mixture in a benchtop microcentrifuge to collect contents at the bottom of the tube.
3. Incubation
• Incubate the mixture at 50 °C for 20 minutes in a dry bath incubator.
Takara Restriction Enzyme Digestion
- Restriction enzyme(s)
- Corresponding reaction buffer
- DNA fragment
- ddH₂O
- Dry bath incubator or incubator
1. Enzyme and buffer selection
• Consult the product catalog to identify the appropriate buffer system and optimal reaction temperature for the chosen enzyme(s).
• For double digestion, confirm buffer compatibility and determine whether a simultaneous (co-digestion) or sequential digestion is required.
2. Reaction setup
• For a 20 μL reaction, add the following components to a PCR tube:
- 1 μL restriction enzyme
- 2 μL of the corresponding 10× buffer
- 1 μg DNA fragment (adjust volume according to concentration)
- ddH₂O to a final volume of 20 μL
3. Digestion
• Incubate the reaction mixture at the recommended temperature in a dry bath incubator or incubator for 6 hours.
4. Post-digestion treatment
• If DNA fragments are to be recovered: heat-inactivate the enzyme at ≥65 °C for 20 minutes, or alternatively proceed directly to DNA purification (e.g., column purification or agarose gel electrophoresis followed by gel extraction).
• If only verification is required: add loading buffer to a final concentration of 1× and analyze by agarose gel electrophoresis.
Fast Restriction Enzyme Digestion
- Restriction enzyme(s)
- Corresponding reaction buffer
- DNA fragment
- ddH₂O
- Dry bath incubator or incubator
1. Enzyme and buffer selection
• Consult the product catalog to confirm the appropriate buffer system and optimal reaction temperature for the selected enzyme(s).
• For double digestion, determine buffer compatibility and decide whether to perform co-digestion or sequential digestion.
2. Reaction setup (20 μL system)
• Add the following components into a PCR tube:
- 1 μL restriction enzyme
- 2 μL of the corresponding 10× buffer
- 1 μg DNA fragment (adjust volume according to DNA concentration)
- ddH₂O to a final volume of 20 μL
3. Mixing
• Mix gently and briefly centrifuge to collect liquid at the bottom.
4. Incubation
• Incubate at 37 °C for 5–10 minutes.
5. Post-digestion treatment
• If DNA fragments are required: heat-inactivate the restriction enzyme at ≥65 °C for 20 minutes, or proceed directly with DNA purification (e.g., liquid recovery or agarose gel electrophoresis followed by gel extraction).
• If only verification is needed: add loading buffer to a final concentration of 1× and analyze by agarose gel electrophoresis.
DNA Gel Extraction (SanPrep Column Kit)
- SanPrep Column DNA Gel Extraction Kit
- Water bath (50 °C)
- Benchtop microcentrifuge
1. Check whether Wash Solution has ethanol added; check if Buffer B2 contains precipitate; preheat the water bath to 50 °C.
2. Excise the agarose gel slice containing the target DNA fragment and weigh it.
3. Add 3–6 volumes of Buffer B2 relative to the gel weight. Incubate at 50 °C for 5–10 minutes until the gel is completely dissolved.
4. For DNA fragments <500 bp, add 1/3 volume of isopropanol relative to the volume of Buffer B2.
5. Transfer the dissolved gel solution into a spin column. Centrifuge at 8,000×g for 30 seconds, discard the flow-through.
6. Add 500 μL Wash Solution, centrifuge at 9,000×g for 30 seconds, discard the flow-through.
7. Repeat Step 6 once.
8. Dry-spin the empty column at 9,000×g for 1 minute to remove residual wash buffer.
9. Place the column into a clean 1.5 mL microcentrifuge tube, add 15–40 μL Elution Buffer directly to the center of the membrane, incubate at room temperature for 1 minute, then centrifuge for 1 minute to elute DNA. Collect and store the DNA solution.
PCR Product Purification (SanPrep Column Kit)
- SanPrep Column PCR Product Purification Kit
- Benchtop microcentrifuge
1. Check whether Wash Solution has ethanol added; check if Buffer B3 contains precipitate; ensure Buffer B3 has isopropanol added.
2. Add 5 volumes of Buffer B3 to the PCR reaction mixture, mix thoroughly.
3. Load the mixture into the spin column, centrifuge at 8,000×g for 30 seconds, discard the flow-through.
4. Add 500 μL Wash Solution, centrifuge at 9,000×g for 30 seconds, discard the flow-through.
5. Repeat Step 4 once.
6. Dry-spin the empty column at 9,000×g for 1 minute to remove residual wash buffer.
7. Place the column into a clean 1.5 mL microcentrifuge tube, add 15–40 μL Elution Buffer directly to the center of the membrane, incubate at room temperature for 1 minute, then centrifuge for 1 minute to elute DNA. Collect and store the purified DNA solution.
Protein Lab
Bacterial Protein Expression & Purification
- LB medium
- M9 medium
- IPTG reagent
- Wash buffer A
- Wash buffer B
- Wash buffer C (imidazole 100–500 mM gradient)
- V-5100 Visible Light Spectrophotometer
- High-speed centrifuge
- SONICS Ultrasonic Homogenizer
- HiTrap Desalting Column / AKTA Protein Purification System
- Microplate Reader
1. Inoculate E. coli BL21(DE3) strain into LB medium and culture overnight at 37°C, 180–200 rpm.
2. Dilute the overnight culture into fresh LB medium containing the appropriate antibiotic at a 1:100 ratio (inoculum : medium).
3. Grow at 37°C, 200 rpm for ~3 hours until the culture reaches OD₆₀₀ = 0.6–0.8.
4. Induce protein expression by adding IPTG stock solution (50 mg/mL) to a final concentration of 1 / 0.8 / 0.5 / 0.3 / 0.1 mM (4.76 / 3.808 / 2.38 / 1.428 / 0.476 mL per L medium). Continue incubation at 16°C, 180 rpm for 12–16 h.
5. Harvest cells by centrifugation at 6,000 rpm, 4°C for 10 min. Collect the pellet (pellets can be stored at 4°C if necessary).
6. Resuspend the pellet in 1/10 volume of buffer using a 5 mL pipette or dropper. Keep samples on ice.
7. Lyse cells by ultrasonication on ice: place the probe ~1 cm below the liquid surface. Use a pulse mode (3 s on / 6 s off) for ~10 min or longer as needed.
8. Centrifuge the lysate at 12,000 rpm, 4°C for 45 min. Discard pellet and retain supernatant.
9. Thaw protein samples on ice. Perform all subsequent purification steps at 4°C on ice.
10. Prepare a Ni-NTA affinity column with 600 µL Ni²⁺ resin beads.
11. Equilibrate the column with 3 mL Buffer B, incubate 3 min, collect flow-through; repeat 3 times.
12. Load protein supernatant in portions (~3 mL each). After each load, incubate 3 min, gently pipette up and down, incubate another 3 min, then collect flow-through. Repeat until all supernatant has been loaded.
13. Wash the column with 4 mL Buffer B, incubate 10 min, collect flow-through; repeat 3 times.
14. Elute bound protein sequentially: add 300 µL Buffer C1, incubate 3 min, collect eluate, repeat 3 times; then switch to Buffer C2, repeat the same steps.
Ultracentrifugation
- Samples to be centrifuged
- Sucrose cushion (if required)
1. Pre-cooling
• Place the rotor in a 4 °C refrigerator overnight to ensure complete pre-cooling before use.
2. Power On
• After preparing the samples, switch on the ultracentrifuge (main power on the right side, ON).
• Wait for the instrument to complete self-checking, then open the sliding chamber lid.
3. Sample Loading and Balancing
• Transfer samples into thin-walled, uncapped Beckman ultracentrifuge tubes.
• Balance tubes to a precision of ±0.01 g.
• Insert tubes symmetrically into the rotor.
4. Rotor Installation
• Align the rotor with the centrifuge spindle and gently lower it vertically into place.
• Turn clockwise slightly to secure, then close the chamber lid.
5. Program Setup
• Input centrifugation parameters:
- Speed (based on rotor and tube specifications, not exceeding their maximum rated speed).
- Time.
- Temperature.
• Confirm that settings comply with rotor and tube safety limits.
6. Running
• Start the vacuum pump; wait until vacuum pressure is below 70 µmHg.
• Press Start to initiate centrifugation.
• Remain nearby until the rotor reaches stable speed.
• If abnormal noise or vibration occurs, press STOP immediately to abort.
7. Completion
• After centrifugation finishes, wait for the rotor to come to a complete stop.
• Release vacuum pressure, then open the chamber lid.
• Carefully remove the rotor and retrieve all tubes and adapters.
8. Shutdown and Cleaning
• Remove the rotor, wipe off condensation, and rinse with tap water if any leakage occurred. Air-dry inverted to prevent corrosion.
• Wipe condensation inside the centrifuge chamber. Keep the lid open until the chamber returns to room temperature and is completely dry.
• Close the lid and switch off the power.
- Always use the correct rotor, tubes, and adapters compatible with the instrument.
- Do not exceed the specified maximum speed of the rotor and tubes.
- Balancing precision is critical to prevent rotor damage and ensure operator safety.
- Operators must receive specific ultracentrifuge safety training.
Dialysis
- Dialysis tubing (pre-activated and suitable MWCO)
- Dialysis buffer (target buffer for exchange)
1. Sample Loading
• Carefully transfer the prepared sample into the pre-activated dialysis tubing.
• Expel any trapped air and seal the tubing securely with clamps.
2. Dialysis Process
• Submerge the dialysis bag into a large volume of dialysis buffer.
• Gently stir or rock the buffer to maintain mixing, avoiding vigorous agitation that may damage the membrane or cause foaming.
3. Buffer Exchange
• Replace the external buffer multiple times according to the experimental design, ensuring sufficient buffer volume and exchange frequency to achieve the desired dialysis efficiency.
4. Sample Recovery
• After dialysis, carefully remove the tubing from the buffer.
• Recover the sample into a clean container.
• Record the final sample volume and concentration.
- Select dialysis tubing with the appropriate molecular weight cutoff (MWCO) based on the sample size.
- Ensure a large buffer-to-sample volume ratio (commonly 100–1000×) for efficient exchange.
- Perform dialysis at 4 °C when working with proteins or other unstable biomolecules to maintain stability.
- Avoid prolonged dialysis that may lead to sample dilution or degradation.
SDS-PAGE (Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis)
- Double distilled water (ddH₂O)
- SDS (sodium dodecyl sulfate)
- APS (ammonium persulfate)
- TEMED (tetramethylethylenediamine)
- Acrylamide/Bisacrylamide solution (Acr-Bis)
- Tris-HCl buffer
- SDS running buffer
- 5× loading buffer
- Protein marker (ladder)
- Coomassie Brilliant Blue solution (or alternative staining reagents such as silver stain, enzyme activity stain)
1. Gel Preparation
• Clean the glass plates and assemble the gel casting system.
• Prepare the separating gel solution (adding TEMed last), mix thoroughly, and pour into the gel cassette. Stop pouring ~3 cm below the top edge.
• Overlay with water to prevent evaporation and ensure a flat gel surface.
2. Stacking Gel Preparation
• Once the separating gel has polymerized, carefully remove the overlay water and blot dry with filter paper.
• Prepare the stacking gel solution, mix well, and pour on top of the separating gel. Insert the comb and allow the gel to polymerize for ~10 min.
• Remove the comb carefully after solidification.
3. Electrophoresis Setup
• Place the gel cassette in the electrophoresis tank and fill with SDS running buffer.
• Load samples into the wells:
- 5 μL protein marker.
- 25 μL of each protein sample (20 μL protein + 5 μL 5× loading buffer, boiled or denatured if required).
4. Electrophoresis Conditions
• Run the gel at 90 V for ~20 min (stacking gel phase).
• Increase to 150 V for ~45 min (resolving gel phase) until the dye front (green marker) reaches the bottom.
5. Staining
• Following electrophoresis, stain the gel with Coomassie Brilliant Blue or alternative methods (e.g., silver staining, activity staining) depending on sensitivity and downstream application.
- Always add APS and TEMED immediately before pouring to ensure proper polymerization.
- Avoid introducing air bubbles when casting gels.
- Ensure equal sample loading for consistent band intensity.
- Use freshly prepared running buffer for best results.
Native-PAGE (Non-denaturing Polyacrylamide Gel Electrophoresis)
- Double distilled water (ddH₂O)
- APS (ammonium persulfate)
- TEMED (tetramethylethylenediamine)
- Acrylamide/Bisacrylamide solution (Acr-Bis)
- Tris-HCl buffer
- Native running buffer
- 4× native loading buffer
- Protein marker (native ladder)
- Coomassie Brilliant Blue (or alternative staining reagents such as silver stain or activity stain)
1. Gel Casting
• Clean the glass plates and assemble the gel casting system.
• Prepare the separating gel according to the molecular weight of the target protein (without SDS). Add TEMED last, mix thoroughly, and immediately pour into the gel cassette. Stop pouring when ~3 cm from the top edge. Overlay with water to seal.
2. Stacking Gel Preparation
• Once the separating gel has polymerized, carefully remove the overlay water.
• Prepare the stacking gel (without SDS), add TEMED last, mix thoroughly, and pour on top of the separating gel. Insert the comb and allow to polymerize for ~10 min. Remove the comb gently once solidified.
3. Buffer Addition
• Fill the inner chamber of the electrophoresis tank with native running buffer.
• Add buffer to the outer chamber to ~1/3 full (too little may disrupt the circuit; too much may reduce efficiency).
4. Sample Preparation and Loading
• Keep all protein samples on ice. Do not boil or add denaturing agents (e.g., SDS, β-mercaptoethanol, DTT).
• Mix protein samples with 4× native loading buffer. Load ~10 μL per well.
• Load 10 μL of native protein marker into the first lane.
5. Electrophoresis
• Place the electrophoresis apparatus on ice to avoid heat generation, which may disrupt protein tertiary and quaternary structure.
• Run the gel at 90 V for ~20 min (stacking gel phase), then increase to 150 V for ~45 min (resolving gel phase) until the dye front (green marker) is visible near the bottom.
6. Staining
• Stain the gel with Coomassie Brilliant Blue, or use silver staining/enzyme activity staining depending on the sensitivity required.
- Native-PAGE separates proteins by size, charge, and conformation, not purely by molecular weight.
- Avoid heat and chemical denaturants throughout the process.
- Always run at low temperature (on ice or in a cold room) to preserve protein activity and structure.
Western Blot
- PVDF membrane
- TBST (TBS + 0.1% Tween-20)
- Primary antibody (1° Ab)
- Secondary antibody (2° Ab)
- Blocking buffer (e.g., 5% non-fat dry milk in TBST)
1. SDS-PAGE
• Perform SDS-PAGE to separate proteins on an unstained gel (do not stain the gel).
2. Membrane Preparation
• Cut the PVDF membrane to match the size of the gel.
• Soak the PVDF membrane and filter papers in transfer buffer until fully equilibrated.
3. Protein Transfer
• Assemble the transfer "sandwich" according to the system used (gel in direct contact with the PVDF membrane, avoiding air bubbles).
• Perform transfer under constant current conditions. Adjust transfer time depending on protein molecular weight.
4. Blocking
• After transfer, incubate the PVDF membrane in 10 mL blocking buffer (e.g., 5% non-fat dry milk in TBST) with gentle shaking for 1 hour at room temperature.
5. Primary Antibody Incubation
• Wash the membrane in TBST three times, 10 min each.
• Incubate the membrane in 10 mL of primary antibody solution at 4°C overnight with gentle agitation.
6. Secondary Antibody Incubation
• Wash the membrane in TBST three times, 10 min each.
• Incubate the membrane in 10 mL of secondary antibody solution at room temperature for 1 hour.
7. Detection
• Wash the membrane in TBST three times, 10 min each.
• Add chemiluminescent substrate (ECL reagent) to cover the membrane surface and remove air bubbles.
• Detect signal using an imaging system.
- For high background, increase washing time or use BSA instead of milk as blocking reagent.
- Transfer efficiency depends on protein size:
- <30 kDa → short transfer (30–45 min)
- 30–100 kDa → ~1 h
- 100 kDa → extended transfer or wet transfer system - Always handle the PVDF membrane with clean tweezers and avoid touching the surface.
Agarose Gel Electrophoresis for Proteins
- Bromophenol blue
- Glycerol
- Agarose
- Tris-HCl
- Sodium acetate
- EDTA
- Coomassie Brilliant Blue (CBB)
- Methanol
- Lactic acid
1. Preparation of Bromophenol Blue Stock Solution
• Dissolve 0.02 g bromophenol blue in 2 mL ddH₂O to obtain a 0.2% bromophenol blue solution.
• Store at 4°C.
2. Preparation of Loading Buffer
• Mix 800 μL glycerol with 1 mL 0.2% bromophenol blue solution.
• Adjust the final volume to 2 mL with ddH₂O.
• The final loading buffer contains 40% glycerol + 0.1% bromophenol blue.
• Store at 4°C.
3. Preparation of Agarose Gel
• Prepare a 0.5% agarose gel in modified TAE buffer (without nucleic acid stain).
4. Sample Preparation and Loading
• Mix protein sample with loading buffer at a 1:1 ratio.
• Load 20 μL per well.
• Running buffer: modified low-acetate TAE (40 mM Tris, 5 mM sodium acetate, 1 mM EDTA).
5. Electrophoresis
• Run the gel at a constant voltage of 10 V for ~10 h.
6. Staining
• Stain the gel overnight at 4°C with Coomassie Brilliant Blue.
7. Destaining
• Destain the gel in a solution containing 40% methanol + 7% lactic acid until clear background is obtained.
- This protocol is suitable for native protein separation under mild conditions (non-denaturing).
- Lower acetate concentration in the buffer minimizes overheating during long electrophoresis.
- Ensure complete destaining for sharp visualization of protein bands.
Assembly Lab
Enzyme Encapsulation
- Dialysis membranes (MWCO 100 kDa and 3 kDa)
- 2% (w/v) sodium bicarbonate
- 1 mmol/L EDTA (pH 8.0)
- Distilled water
- Dialysis buffer
(1) Activation of Dialysis Membranes
1. Cut the dialysis tubing into small pieces of appropriate length.
2. Boil the dialysis membranes in a large volume of 2% (w/v) sodium bicarbonate and 1 mmol/L EDTA (pH 8.0) solution for 10 min.
3. Wash thoroughly with distilled water.
4. Boil again in 1 mmol/L EDTA (pH 8.0) solution for 10 min.
5. After cooling, wearing clean gloves, fill the membrane with water and then discard it to remove residual reagents. Rinse thoroughly.
(2) Enzyme Encapsulation Procedure
1. Mix 6 M guanidine hydrochloride (GuHCl) with 0.5 mg/mL CP-ST at a 1:1 volume ratio to obtain a final 3 M GuHCl solution. Incubate on ice for 1 h.
2. Add an appropriate volume of SlPPK-SP to the mixture so that the final GuHCl concentration is 1.5 M, with a molar ratio of SlPPK-SP : CP-ST = 1:2.
3. Transfer the mixture into the pre-activated 3 kDa dialysis tubing, and dialyze against 100× volume dialysis buffer at 4°C for 12–18 h.
4. Transfer the dialyzed solution into the pre-activated 100 kDa dialysis tubing, and dialyze against 100× volume dialysis buffer at 4°C for 12–18 h to remove unassembled free proteins.
- Activation of dialysis tubing ensures removal of preservatives and improves permeability.
- Stepwise reduction of GuHCl concentration promotes proper folding and assembly of enzyme complexes.
- The second dialysis step with higher MWCO tubing allows removal of free unassembled proteins while retaining encapsulated complexes.
Size-Exclusion Chromatography (SEC)
- Purified water
- Buffer B
- Absolute ethanol
- Protein sample solution
1. Gel Selection
• Select a gel matrix with an appropriate fractionation range based on the molecular weight, resolution requirements of the target protein and impurities, and sample stability.
2. Gel Swelling
• Dry gel must be fully swollen before use (gel : water = 1:10).
• Add purified water or Buffer B to the dry gel in a beaker, stir thoroughly, and let it stand.
• Discard the supernatant suspension, remove gel fragments floating on the surface, and repeat this process until the supernatant becomes clear.
3. Column Dimensions
• Determine the required bed volume, length, diameter, and aspect ratio of the gel column based on the sample volume, sample properties, and desired separation resolution.
4. Column Packing
• Close the outlet of the column and add ~1/3 of the total bed volume of water or buffer.
• Resuspend the pre-swollen gel in buffer, and gently pour the slurry into the column in one continuous step along the column wall.
• When the gel settles to ~5 cm in height, open the column outlet and adjust the flow rate to ~1 mL/min to allow uniform packing.
5. Sample Loading
• Use an appropriate buffer as the mobile phase.
• The sample volume should generally be 1–5% of the total column bed volume.
• Prior to loading, filter (0.22 µm) or centrifuge the sample to remove particulates that could clog the column.
6. Elution
• Elute with either a single buffer system or a buffer containing salt, depending on experimental requirements.
• Maintain a stable operating pressure and ensure the flow rate does not exceed 0.5–3.0 mL/min.
- Avoid air bubbles in the column; always degas the buffer before use;
- Never allow the column bed to dry, keep it constantly hydrated;
- Control sample concentration and injection volume to prevent overloading and loss of resolution;
- Monitor system pressure during operation to avoid column damage due to overpressure.
High-Performance Liquid Chromatography (HPLC)
- Potassium dihydrogen phosphate (KH₂PO₄)
- Methanol (HPLC grade)
- Ultrapure water
- HPLC system equipped with a UV detector
- C18 reversed-phase column (4.6 × 150 mm, Wondasil, Shimadzu GL Sciences)
- Dionex Chromeleon software for data acquisition and analysis
1. Chromatographic Conditions
• For general nucleotide detection:
- Mobile phase: 50 mM potassium dihydrogen phosphate : methanol (v/v 97:3)
- Flow rate: 0.6 mL/min
- Column temperature: 30 °C
- Detection wavelength: 270 nm
• For adenosine detection:
- Mobile phase: 50 mM potassium dihydrogen phosphate : methanol (v/v 90:10)
- Flow rate: 0.8 mL/min
- Column temperature: 30 °C
- Detection wavelength: 270 nm
2. Sample Preparation
• Dilute samples with ultrapure water.
• Filter through a 0.22 μm hydrophilic PTFE syringe filter (Anpel, Shanghai).
• Inject 10 μL of the filtrate for HPLC analysis.
3. Data Analysis
• Chromatographic data were recorded and processed using Dionex Chromeleon software.
- All solvents must be freshly prepared and filtered through a 0.22 μm membrane to avoid baseline noise and column clogging.
- Equilibrate the column with the mobile phase for at least 20 minutes before sample injection.
- Use an appropriate standard curve for quantification of analytes.
Transmission Electron Microscopy (TEM)
- Prepared samples
- 300-mesh carbon support film
- TEM instrument with operation software
1. Turn on the TEM system sequentially: main power → vacuum pump → cooling system → high-voltage power supply. Wait until the self-check process is completed, then enter the operation software.
2. Carefully mount the prepared sample on a 300-mesh carbon support film, fix it onto the loading rod, and insert into the TEM sample holder.
3. Set appropriate imaging parameters, including acceleration voltage, magnification, and focusing mode.
4. Activate the electron beam. Adjust the stage position, focusing rings, and apertures until a clear image is obtained. Acquire images and transfer the data to a computer for analysis.
5. After imaging, sequentially switch off the electron beam, high-voltage power supply, vacuum pump, cooling system, and main power supply.
Notes:- Samples should be properly prepared, dried, and evenly distributed on the carbon film to avoid charging effects.
- Avoid prolonged exposure of the same area to the electron beam to minimize radiation damage.
- Ensure stable vacuum conditions throughout operation for optimal imaging quality.
- Only trained personnel should operate the TEM, due to high-voltage and vacuum safety concerns.
Dynamic Light Scattering (DLS)
- Pure/deionized water (same solvent as sample)
- Disposable plastic or quartz cuvettes
- Protein, nanoparticle, or colloid samples
1. Turn on the DLS instrument, allowing the laser and detector to preheat and stabilize (typically 30 min).
2. Clean the sample chamber and cuvette with pure solvent. Avoid fingerprints and dust; if necessary, blow dry with filtered nitrogen.
3. If reusing cuvettes, wash with detergent, rinse thoroughly with deionized water, followed by filtered water, then rinse with solvent and dry with lint-free paper or air-dry.
4. Prepare the sample: dissolve in compatible buffer/solvent to obtain a clear solution. Typical protein concentration is 0.1–2 mg/mL; nanoparticles should follow manufacturer's or pre-experimental guidelines.
5. Remove aggregates and bubbles: centrifuge samples at 12,000–20,000 × g for 5–15 min or filter through 0.22 μm membranes (if compatible with particle size).
6. Load 1–2 mL of sample into a clean cuvette, avoiding bubbles. Seal with a cap.
7. Place the cuvette into the instrument and ensure proper alignment. For temperature-controlled measurements, set the desired temperature and wait 2–5 min for equilibration.
8. Input sample parameters into the software, including solvent refractive index, viscosity, and measurement wavelength (or allow software auto-calculation).
9. Configure measurement settings:
• Number of runs: 3–12 repeats (commonly 5)
• Acquisition time: 10–60 s per run
• Ensure the count rate falls within the optimal detection range.
10. Start data acquisition and perform multiple repeats per sample to ensure reproducibility.
11. Save results, analyze data, and plot size distribution (log10 particle diameter in nm vs. intensity).
Notes:- Samples must be transparent and free of dust or bubbles to avoid scattering artifacts.
- Protein and nanoparticle concentrations should be optimized to prevent multiple scattering or low signal.
- Always use solvent-matched blanks for calibration.
- Avoid vibration or strong light interference during measurement.
Pull-down Assay
- Protein A/G Magnetic Magnetic Beads
- Binding Buffer
- Elution Buffer
- FLAG antibody (or other tag-specific antibody)
- 1.5 mL microcentrifuge tubes
1. Vortex the Protein A/G Magnetic Beads for 30 s to ensure they are well resuspended.
2. Transfer 50 μL of beads into a 1.5 mL microcentrifuge tube and place on a magnetic stand to separate.
3. Add 150 μL of binding buffer, gently resuspend, and separate using the magnet. Repeat this washing step twice.
4. Dilute 10 μL of antibody in 40 μL binding buffer.
5. Incubate the diluted antibody with the magnetic beads at room temperature for 1 h on a rotator.
6. After incubation, gently resuspend and then separate using the magnet.
7. Wash beads with 150 μL binding buffer, gently resuspend, and separate with the magnet.
8. Add 100 μL of protein sample to the beads, gently resuspend, and incubate at room temperature for 10 min.
9. After incubation, gently resuspend and separate with the magnet.
10. Wash the beads with 300 μL binding buffer, separate with the magnet, and repeat the washing step three times.
11. Add 150 μL binding buffer, transfer the beads to a new microcentrifuge tube, and separate with the magnet.
12. Elute bound proteins by adding 100 μL of elution buffer, resuspending, and separating. Collect the eluate for downstream analysis (e.g., SDS-PAGE, Western blot, or mass spectrometry).
Notes:- Always keep beads in suspension by gentle mixing to maximize binding efficiency.
- Avoid introducing bubbles during pipetting, which may reduce reproducibility.
- Use freshly prepared binding and elution buffers for optimal results.
- For weak interactions, consider longer incubation times or lower stringency wash buffers.
- Work quickly at low temperature (e.g., 4 °C) if target proteins are unstable.
Agarose-based ELISA
- Buffer B
- 75% ethanol
- Agarose (1% solution prepared fresh)
- 5% skim milk (blocking solution)
- Primary antibody
- Secondary antibody
- ECL substrate (chemiluminescent detection reagent)
- Glass slides
- Agarose beads (400 μL per assay)
1. Clean glass slides with 75% ethanol, allow to air dry.
2. Take 400 μL beads and wash 3 times with Buffer B.
3. Prepare 15 mL of 1% agarose with Buffer B, maintain in a melted state.
4. Mix 100 μL melted agarose with 100 μL beads immediately, and spread evenly on the slide using a pipette tip. The agarose layer should form a continuous thin coating on the slide surface, without breaks or aggregates.
5. Place slides at 4 °C to allow agarose to solidify.
6. Wash slides 3 times with Buffer B.
7. Apply 100 μL of sample to the agarose-coated slide, incubate at room temperature for 30 min.
8. Wash slides 3 times with Buffer B.
9. Block the slide by immersing it in 5% skim milk for 30 min at room temperature.
10. Wash slides 3 times with Buffer B.
11. Incubate slides with primary antibody solution for 1 h at room temperature.
12. Wash slides 3 times with Buffer B.
13. Incubate slides with secondary antibody for 1 h at room temperature.
14. Wash slides 3 times with Buffer B.
15. Develop signal using ECL substrate, and detect chemiluminescence with an imaging system.
Notes:- The agarose coating must be continuous and uniform; uneven or cracked gels will cause inconsistent signal.
- Keep agarose solution melted before mixing with beads; solidification during handling will reduce reproducibility.
- Blocking with 5% skim milk is critical to minimize non-specific binding.
- All washes should be performed gently to avoid detaching the agarose layer.
- Perform ECL development in the dark and image promptly to prevent signal decay.
Dot Blotting
- Methanol
- Transfer buffer
- TBST (Tris-buffered saline with 0.1% Tween-20)
- 5% skim milk (blocking buffer)
- Primary antibody
- Secondary antibody
- ECL substrate
1. Membrane activation
• Immerse the PVDF membrane in methanol for 1 min.
• Wash thoroughly in transfer buffer to remove methanol.
• Place the membrane on a flat surface and allow residual water to air-dry naturally (do not over-dry).
2. Sample loading
• Spot 1–3 μL of sample solution directly onto the membrane surface.
• Allow the membrane to absorb the sample completely (avoid forced air-drying).
3. Washing & blocking
• Wash the membrane twice with TBST for 5 min each.
• Block with 5% skim milk in TBST at room temperature with gentle shaking for 40 min.
4. Primary antibody incubation
• Wash the membrane 3 times with TBST (10 min each).
• Incubate the membrane with diluted primary antibody at 4 °C overnight with gentle shaking.
5. Secondary antibody incubation
• Wash the membrane 3 times with TBST (10 min each).
• Incubate with the corresponding secondary antibody for 1 h at room temperature.
6. Detection
• Wash the membrane with TBST (3 × 10 min).
• Add ECL substrate evenly over the spotted areas, avoid bubbles.
• Detect the chemiluminescent signal using an imaging system.
- Avoid applying excessive sample volume, as it can cause diffusion and blurred spots.
- Ensure equal sample volume for each spot to enable semi-quantitative comparison.
- If background is high, extend washing steps or use BSA as an alternative blocking agent.
- Membrane should not be allowed to dry completely after sample spotting until blocking is finished.
GSH ELISA
- Methanol
- Transfer buffer
- TBST (Tris-buffered saline with 0.1% Tween-20)
- 5% skim milk (blocking buffer)
- Primary antibody
- Secondary antibody
- ECL substrate
1. Membrane activation
• Immerse the PVDF membrane in methanol for 1 min.
• Wash thoroughly in transfer buffer to remove methanol.
• Place the membrane on a flat surface and allow residual water to air-dry naturally (do not over-dry).
2. Sample loading
• Spot 1–3 μL of sample solution directly onto the membrane surface.
• Allow the membrane to absorb the sample completely (avoid forced air-drying).
3. Washing & blocking
• Wash the membrane twice with TBST for 5 min each.
• Block with 5% skim milk in TBST at room temperature with gentle shaking for 40 min.
4. Primary antibody incubation
• Wash the membrane 3 times with TBST (10 min each).
• Incubate the membrane with diluted primary antibody at 4 °C overnight with gentle shaking.
5. Secondary antibody incubation
• Wash the membrane 3 times with TBST (10 min each).
• Incubate with the corresponding secondary antibody for 1 h at room temperature.
6. Detection
• Wash the membrane with TBST (3 × 10 min).
• Add ECL substrate evenly over the spotted areas, avoid bubbles.
• Detect the chemiluminescent signal using an imaging system.
- Avoid applying excessive sample volume, as it can cause diffusion and blurred spots.
- Ensure equal sample volume for each spot to enable semi-quantitative comparison.
- If background is high, extend washing steps or use BSA as an alternative blocking agent.
- Membrane should not be allowed to dry completely after sample spotting until blocking is finished.
Temperature-Dependent Assay
- ATP detection reagent
- Dilution buffer
- Standard substrate solution
- Distilled water
- Enzyme sample
- Assembled protein/sample mixture
- Microplate reader
- 96-well microplate
1. Preparation of ATP working solution
• Mix 10 μL ATP detection reagent with 90 μL dilution buffer.
• Prepare 100 μL of ATP working solution per reaction group.
2. Loading substrate
• Add 100 μL ATP working solution to each well of the microplate.
• Incubate at room temperature for 2 min.
• Add 20 μL standard substrate solution to each well.
3. Enzyme treatment
• Pre-incubate enzyme samples under the desired temperature conditions.
• Heat in a metal bath for 10 min, then immediately transfer.
• Add 10 μL pre-treated enzyme sample to each well.
4. Bioluminescence measurement
• Mix briefly in the microplate reader (2 s).
• Record bioluminescence intensity every 20 s for a total of 15 min.
• Export data and plot bioluminescence vs. time curves for analysis.
- Ensure all reagents are equilibrated to room temperature before use.
- Maintain consistent pipetting speed and timing to minimize variability between wells.
- Optimize excitation/emission wavelengths of the plate reader according to the ATP detection kit specifications.
- For comparative assays, process all samples in parallel to ensure uniform incubation times.
pH-Modulation Assay
- ATP detection reagent
- Dilution buffer
- Standard substrate solution
- 1 M HCl (for pH adjustment)
- Distilled water
- Enzyme sample
- Assembled protein/sample mixture
- Microplate reader
- 96-well microplate
1. Preparation of ATP working solution
• Mix 10 μL ATP detection reagent with 90 μL dilution buffer.
• Prepare 100 μL of ATP working solution per reaction group.
2. Loading substrate
• Add 100 μL ATP working solution to each well of the microplate.
• Incubate at room temperature for 2 min.
• Add 20 μL standard substrate solution to each well.
3. Enzyme and pH gradient treatment
• Add 10 μL enzyme sample to each well and mix gently by pipetting.
• Immediately add 30 μL of pre-prepared gradient pH buffer B solution to adjust reaction conditions.
4. Bioluminescence measurement
• Mix in the microplate reader for 5 s.
• Record bioluminescence intensity every 30 s for a total of 15 min.
• Save raw data and generate plots of bioluminescence vs. time under different pH conditions.
- Ensure all pH buffer solutions are freshly prepared and equilibrated to room temperature before use.
- Handle 1 M HCl with caution; always wear appropriate PPE.
- To minimize variability, prepare all pH gradient conditions in parallel.
- Optimize excitation/emission wavelengths of the plate reader according to the ATP detection kit specifications.
Phosphate Substrate Inhibition Assay
- ATP detection reagent
- Standard substrate solution
- Dilution buffer
- Phosphate solution (prepared at different gradient concentrations)
- Distilled water
- Enzyme sample
- Assembled protein/sample mixture
- Microplate reader
- 96-well microplate
1. Preparation of ATP working solution
• Mix 10 μL ATP detection reagent with 90 μL dilution buffer.
• Prepare 100 μL ATP working solution per reaction group.
2. Loading substrate
• Add 100 μL ATP working solution to each well of the microplate.
• Incubate at room temperature for 2 min.
• Add 20 μL standard substrate solution to each well.
3. Enzyme and phosphate gradient treatment
• Add 10 μL enzyme sample to each well and mix gently by pipetting.
• Immediately add phosphate solution at different gradient concentrations, corresponding to 10% of the total reaction volume.
4. Bioluminescence measurement
• Mix in the microplate reader for 5 s.
• Record Bioluminescence intensity every 30 s for a total of 15 min.
• Save raw data and generate inhibition curves by plotting relative Bioluminescence vs. phosphate concentration.
- Phosphate solutions should be freshly prepared to avoid precipitation and pH drift.
- Optimize excitation/emission wavelengths of the plate reader according to the ATP detection kit specifications.
- Perform at least three biological replicates for reliable kinetic analysis.
Equipment
Use of Micropipette
1. Attach the appropriate tip to the pipette.
2. Adjust the pipette volume to the required value.
3. Hold the pipette in the air and press it down to the first stop.
4. Immerse the pipette tip below the surface of the liquid to be aspirated.
5. Slowly release the plunger to the original position.
6. Move the pipette to the target container.
7. Press the plunger down to the second stop to dispense all the liquid.
8. Press the tip ejector button to discard the tip.
Reverse Pipetting (Press 2 – Release 1)1. Attach the appropriate tip to the pipette.
2. Adjust the pipette volume to the required value.
3. Hold the pipette in the air and press it down to the second stop.
4. Immerse the pipette tip below the surface of the liquid to be aspirated.
5. Slowly release the plunger to the original position.
6. Move the pipette to the target container.
7. Press the plunger down to the first stop to dispense the required volume of liquid.
8. Press the tip ejector button to discard the tip.
Notes:1. Each pipette corresponds to specific tips. Different volume ranges require different tips, which are often color-coded.
2. Always use the pipette within its specified range; do not go below the minimum volume.
3. When attaching the tip, apply firm pressure and gently rock side to side to ensure it is securely fitted.
4. Forward pipetting is used in most pipetting situations. Reverse pipetting is suitable for viscous liquids (where forward pipetting may leave residue and cause inaccurate pipetting) or when bubbles must be avoided (since forward pipetting can easily generate bubblesbubbles).
5. When using reverse pipetting, aspirate the liquid slowly; otherwise, due to inertia, the liquid may surge upwards and contaminate the pipette.
6. Do not place the pipette horizontally or upside down after aspirating liquid, to prevent contamination.
7. After use, set the pipette to its maximum volume and place it back in its holder.
8. Tips should be sterilized using autoclaving at 121°C for 20 minutes. Secure the tip box with rubber bands, and wrap in kraft paper or newspaper if necessary.
Tray Balance
Used to weigh solid or liquid samples required for experiments, ensuring accurate measurement.
Materials:- Sample to be weighed
- Weighing paper or weighing boat
1. Before use, check whether the balance is level and adjust the leveling screws if necessary;
2. Ensure the sliding weight is set to zero and the pointer aligns with the zero mark on the scale;
3. Place the weighing paper or weighing boat on the pan and re-balance to zero;
4. Gradually add the sample to the weighing paper or boat until it approaches the desired mass;
5. Move the sliding weight to fine-tune until the pointer returns to zero, then record the reading;
6. After weighing, clean the weighing paper and remove any residual sample to keep the balance clean;
7. When finished, return the sliding weight to zero and cover the balance with the dust cover.
Electronic Balance
Used for rapid and precise weighing of solid or liquid samples required for experiments.
Materials:- Sample to be weighed
- Weighing paper or weighing boat
1. Before use, check that the electronic balance is placed in a stable environment free of vibration and airflow;
2. Turn on the power and allow the balance to preheat for a few minutes to ensure stable readings;
3. Place the weighing paper or weighing boat on the pan and press the TARE button to reset the display to zero;
4. Slowly add the sample to avoid spillage or overloading;
5. Read the mass value displayed on the screen and record it promptly;
6. After weighing, remove the sample and confirm the balance returns to zero;
7. After use, turn off the power and keep the balance clean to prevent residues from affecting accuracy.
Electronic Analytical Balance
Used for high-precision measurement of sample mass, suitable for scientific experiments that require high weighing accuracy.
Materials:- Sample to be weighed
- Weighing paper or weighing boat
- Tweezers (for placing or removing samples to avoid contamination by hand contact)
1. Place the balance in a stable environment free of vibration, airflow, and with constant temperature. Ensure it is properly leveled;
2. Turn on the power and allow the balance to preheat for about 30 minutes to ensure measurement stability;
3. Open the draft shield, place the weighing paper or weighing boat on the pan, close the draft shield, and press the TARE button to reset to zero;
4. Use tweezers to gently place the sample into the weighing paper or boat, close the draft shield, and wait for the reading to stabilize;
5. Record the mass value displayed on the screen promptly;
6. For multiple measurements, press the TARE button to zero before each weighing;
7. After finishing, remove the weighing paper or boat, close the draft shield, turn off the power, and keep the balance clean;
8. Do not overload the balance to avoid damaging the sensor.
Mini Centrifuge
Used for rapid sedimentation or separation of small-volume samples (such as PCR tubes or microcentrifuge tubes).
Materials:- Microcentrifuge tubes or PCR tubes
- Sample solution
1. Before use, check that the power supply is normal and ensure the centrifuge is placed on a stable laboratory bench;
2. Add the sample into the centrifuge tube, ensuring that the number of tubes used is symmetrical and the mass is balanced;
3. Open the centrifuge lid, place the tubes into the rotor symmetrically;
4. Close the lid securely and start centrifugation. Do not open the lid during operation. Remove the samples only after the rotor has completely stopped;
5. After use, turn off the power, wipe the surface of the centrifuge, and keep it clean;
6. Regularly check the rotor for cracks or wear to avoid potential safety hazards.
Mini Benchtop Centrifuge
Used for sedimentation and separation of small-volume samples (such as cells, proteins, or nucleic acid solutions).
Materials:- Microcentrifuge tubes or compatible sample tubes
- Sample solution to be separated
1. Before use, ensure the centrifuge is placed on a stable and level laboratory bench;
2. Add samples into centrifuge tubes, ensuring strict balance between tubes;
3. Open the centrifuge lid and place the tubes symmetrically into the rotor;
4. Close the lid securely and set the appropriate speed and centrifugation time;
5. Start the centrifuge. Do not open the lid during operation;
6. Wait until the rotor has completely stopped before opening the lid and removing samples;
7. After use, turn off the power, wipe the inner chamber and outer surface of the centrifuge to remove condensation or residues;
8. Regularly inspect the rotor and tube adapters for cracksss or damage to avoid safety risksss.
High-Speed Centrifuge
Used to collect cells or insoluble fractions after cell lysis by centrifugation.
Materials:- Bacterial solution after induction of expression
1. Strictly ensure balance!
2. The centrifuge rotor must be used together with the corresponding lid and centrifuge containers;
3. The rotor and lid must be tightened securely;
4. Centrifugation parameters must comply with the rotor’s usage requirements;
5. The loading volume must not exceed 2/3 of the container capacity;
6. After use, wipe the centrifuge to remove condensation, and store the rotor inverted in a 4 °C refrigerator.
Ultracentrifuge
Used for high-speed separation of proteins, nucleic acids, viral particles, organelles, and other biomolecules. It is suitable for ultrahigh-speed sedimentation and separation of substances with small molecular weight or close density.
Materials:- Sample solution to be separated
- Specialized ultracentrifuge tubes
- Matching rotor and rotor lid
1. Strictly balance samples: Centrifuge tubes must be placed in pairs with weight differences controlled within 0.001 g;
2. Use specialized tubes: Ensure centrifuge tubes match the rotor model to prevent rupture under high centrifugal force;
3. Sample loading: Fill centrifuge tubes completely to avoid cavitation. Use sealing caps or sealing film if necessary;
4. Install rotor: Place the rotor into the chamber, ensure it is stable, and tighten the rotor lid securely;
5. Set parameters: Adjust speed and time according to experimental requirements, strictly following the rotor’s maximum speed and temperature limits;
6. Safety check: Close the centrifuge chamber lid, ensure the chamber is properly vacuum-sealed before starting;
7. Do not open lid during operation: The centrifuge must not be opened while running. The operator should stay in the lab and monitor the process;
8. Finishing operation: Wait until the rotor has completely stopped before opening the lid and removing samples. Do not force stop the centrifuge;
9. Maintenance: After use, wipe condensation from the chamber, store the rotor inverted in a 4 °C refrigerator to prevent corrosion and damage;
10. Notes: Only trained personnel are allowed to operate the ultracentrifuge. Untrained individuals are strictly prohibited from using it.
Vortex Mixer
Used for rapid mixingiing of small-volume liquid sampleses, such as solutions, suspensionss, and reagents.
Materials:- Sample tubes (e.g., EP tubes, centrifuge tubeses)
- Samples to be mixed
1. Before use, check that the instrument is placed stably on the laboratory bench;
2. Turn on the power switch and select the appropriate mode (touch mode or continuous mode);
3. Hold the bottom of the sample tube vertically and press it gently onto the mixingiing head, ensuring full contact;
4. Adjust the speed control knob to select the appropriate vortexing speed as required;
5. Avoid applying excessive force during mixingiing to prevent tube damage or liquid splashing;
6. After mixingiing, remove the sample tube and turn off the power;
7. After use, keep the instrument clean and avoid reagent residues on the mixiing head.
Magnetic Stirrer
Used for uniform stirring and mixing of liquid samples, commonly applied in dissolving, reactions, or maintaining solution homogeneity.
Materials:- Solution to be stirred
- Beaker or Erlenmeyer flask
- Stir bar (magnetic bar)
1. Before use, ensure the stirrer is placed on a level and stable laboratory bench;
2. Place an appropriately sized stir bar into the container, then add the solution to be stirred;
3. Position the container at the center of the magnetic stirrer, aligning it with the stir bar;
4. Turn on the power and slowly adjust the speed control knob, gradually reaching the desired stirring speed to avoid stir bar jumping or liquid splashing;
5. If heating is required, turn on the heating function and set the desired temperature, taking care to avoid overheating;
6. Monitor the process to prevent excessive evaporation or container movement;
7. After stirring, reduce the speed to zero before turning off the power;
8. Remove the stir bar, clean the container and the instrument, and keep the device in good condition.
Water Bath
Used for constant temperature heating of samples, enzyme reactions, incubation of cells or proteins, and other laboratory temperature control operations.
Materials:- Samples to be heated or kept at constant temperature
- Suitable containers (e.g., centrifuge tubes, culture flasks)
- Distilled water or deionized water (for filling the water bath)
1. Before use, check whether there is sufficient water in the bath. The water level should be above the heating element to avoid dry heating;
2. Connect the power supply and turn on the power switch;
3. Set the required temperature and wait until the water bath reaches and stabilizes at the set temperature;
4. Place the samples in appropriate containers and immerse them in the water bath, ensuring proper sealing to avoid water vapor contamination;
5. During the experiment, regularly check whether the temperature is stable and the water level is sufficient;
6. After the experiment, remove the samples and turn off the power switch;
7. If not used for a long time, drain the water from the bath and wipe it clean to keep the equipment dry and tidy.
Dry Bath
Used for constant temperature heating of samples, enzyme reactions, nucleic acid denaturation/renaturation, protein incubation, and other experimental operations. Commonly used as a liquid-free alternative to a water bath.
Materials:- Samples to be heated
- Dedicated centrifuge tubes or PCR tubes
- Corresponding metal heating blocks (different apertures match different tube types)
1. Before use, ensure the dry bath is placed stably and the power supply is properly connected;
2. Install the appropriate metal block (ensure it matcheshes the type of tube used in the experiment);
3. Turn on the power switch, set the required temperature and time, and wait until the bath reaches and stabilizes at the set value;
4. Place the sample tubeses into the holeses of the metal block, ensuring close contact for uniform heat transfer;
5. During heating, avoid frequent opening of the cover or moving the sampleses to maintain temperature stability;
6. After the experiment, remove the sampleses and turn off the power;
7. Once cooled, wipe the surface of the metal block to keep it clean and avoid corrosion or contamination.
SONICS Ultrasonic Cell Disruptor
Used to disrupt bacteria for protein extraction.
Materials:- Bacterial suspension resuspended in protein buffer
- Ultrapure water
1. Avoid running without load (the ultrasonic probe must be inserted into the sample solution before starting ultrasound);
2. The insertion depth of the ultrasonic probe should be 1/3–2/3 of the liquid level. Insertion that is too deep will reduce disruption efficiency. The probe should be positioned in the center of the liquid and must not touch the container wall;
3. Different probes correspond to different ultrasonic power levels;
4. Set the amplitude using the “AMPL” button;
5. Set the pulse duration for each ultrasound using the “PULSER” button (Pulse on = ultrasound; Pulse off = pause);
6. Set the total ultrasound time using the “TIMER” button:
o Total time = time per pulse × number of pulses.
Alcohol Burner
1. Open the alcohol burner cover and light the burner using a lighter or a match.
2. To extinguish the alcohol burner, cover it twice with the cap to ensure the flame is completely out.
Notes:1. Before using the alcohol burner, check whether there is sufficient alcohol inside to avoid burning the wick when empty.
2. The alcohol burner should be filled with 95% ethanol.
3. Before lighting, make sure there is no alcohol or other flammable liquid on gloves or the workbench.
4. When sterilizing instruments (such as inoculation loops or needles), use the outer flame of the alcohol lamp.
Autoclave
1. Check the external water level gauge of the autoclave to ensure the water level is neither too high nor too low. Check the pressure gauge to confirm normal pressure. If the water level is too high, use the drain pipe at the bottom to release water until the level is appropriate.
2. Turn on the autoclave power and switch, and after confirming safety, open the lid.
3. Check the water level inside the autoclave to ensure it is not too high or too low. If too low, add distilled water.
4. Place the items to be sterilized in heat-resistant containers.
5. Close the lid, confirm it is sealed properly, then set the parameters and start sterilization.
6. After sterilization, wait until the pressure is safe and the temperature drops below 60 °C before opening the lid to remove sterilized items.
7. Tighten the lid of the autoclave and turn off the switch and power supply.
Notes:1. When opening the lid, always press down on the center of the lid with one hand. Do not press the left or right side.
2. Once the lid is opened, hold it with both hands. When closing, also support the lid with both hands.
3. Do not add tap water or other ion-rich water, as it can cause scale buildup and localized overheating. Only distilled water should be added.
4. Sterilized items should not be stacked too high or touch the autoclave lid.
5. For LB medium, typically sterilize at 121 °C for 20 minutes. For glucose-containing medium, do not sterilize at 121 °C (to avoid caramelization). Instead, filter-sterilize glucose solutions. Antibiotics decompose at high temperature and should also be sterilized by filtration. For reagentsss that require sterilization, heating, or dissolution, select the corresponding program.
6. For glassware, do not seal completely. Use sealing film to loosely cover openings. For test tubeses, do not screw caps on tightly. For reagent bottles, also avoid completely tightening caps.
7. For pipette tip sterilization, secure the tip boxesess with rubber bands, and wrap them with newspaper or kraft paper if necessary.
8. For glassware, pipette tips, waste liquidsss, and used culture media, select 121 °C for 20 minutes.
9. Used culture media should be placed in plastic trash bags before sterilization.
10. After starting sterilization, you must sign the log sheet. Ensure the log sheet corresponds exactly to the specific autoclave being used.
11. After sterilization, only open the lid when the temperature has dropped below 60 °C, and be careful of steam when opening.
12. After using the autoclave, check whether any items remain inside. Confirm all have been removed before closing the lid securely.
Laminar Flow Clean Bench
Provides a sterile, dust-free localized clean environment for sample handling. Commonly used in cell culture, aseptic operations, and molecular biology experiments.
Materials:- Experimental samples to be handled
- Sterile consumables (e.g., pipettes, tips, Petri dishes, centrifuge tubes)
- 75% ethanol or other disinfectants
1. Preparation before use
o Turn on the clean bench power and fan, and let it run for 15–30 minutes to establish stable laminar flow;
o Wipe the work surface and item surfaces with 75% ethanol to maintain sterility.
2. Arrangement of items
o Place required items in the bench according to the order of use, avoiding blockage of airflow;
o During operation, minimize crossing movements of hands and items inside the bench to avoid disturbing laminar flow.
3. Experimental operation
o Operators must wear lab coats, masks, and disposable gloves;
o Perform all manipulations in the central area of the airflow;
o Avoid sudden or large movements to maintain laminar flow stability.
4. Post-use procedure
o Remove samples and consumables after the experiment;
o Wipe the work surface again with 75% ethanol;
o Turn off the fan and power. If conditions allow, turn on the UV lamp for 15–30 minutes for disinfection.
5. Notes
o A laminar flow clean bench only protects the sample from contamination, but does not protect the operator. For work involving pathogens or hazardous gases, a biosafety cabinet must be used instead.
Incubator
Used for constant-temperature culture of cells, microorganisms, or tissue samples, providing a stable temperature and environmental conditions.
Materials:- Samples to be cultured (e.g., cell culture flasks, Petri dishes, culture media)
- Suitable culture containers
- Disinfectants (e.g., 75% ethanol)
1. Preparation before use
o Check that the incubator is placed stably and that the power connection is normal;
o Wipe the inside of the incubator with 75% ethanol to maintain cleanliness;
o Set the temperature according to experimental requirements (e.g., 37 °C), and wait until it stabilizes.
2. Placing samples
o Place culture samples in the incubator, avoiding overcrowding of containers to ensure even airflow and temperature distribution;
o For CO₂ incubators, confirm that gas supply is functioning properly, and maintain appropriate humidity to prevent evaporation of culture medium.
3. During incubation
o Minimize the frequency and duration of opening the incubator door to avoid fluctuations in temperature, humidity, and gas concentration;
o Regularly monitor the culture status, and if necessary, perform handling under aseptic conditions.
4. After use
o Remove samples and clean up any remaining containers after the experiment;
o Regularly disinfect and clean the incubator to prevent microbial contamination;
o If not in use for a long time, turn off the power and keep the incubator dry.
5. Notes
o Different types of incubation require suitable incubators (e.g., standard constant-temperature incubator, CO₂ incubator, shaking incubator);
o Do not place flammable, volatile, or toxic substances inside.
Shaker
Used for mixing and culturing liquid samples, commonly applied in cell culture, microbial culture, and accelerating solution reactions.
Materials:- Samples to be cultured or mixed (e.g., Erlenmeyer flasks, culture flasks, centrifuge tubes)
- Culture medium or reaction solution
1. Preparation before use
o Check that the shaker is placed stably and the power connection is normal;
o Ensure that clamps or the platform are securely installed to prevent samples from sliding during operation.
2. Placing samples
o Place sample containers evenly on the shaker platform to maintain balance and avoid uneven loading;
o Use appropriate clamps or rubber bands to secure containers and prevent them from falling during operation.
3. Setting parameters
o Set the shaking speed (rpm) and running time according to experimental requirements;
o For temperature-controlled shakers, also set the required temperature and wait until stabilization before starting the experiment.
4. During operation
o Start the shaker and ensure smooth operation without abnormal noise;
o Avoid opening the lid or moving samples during operation;
o Regularly monitor the culture or mixing process.
5. After use
o Stop the shaker, and wait until it has completely come to rest before removing samples;
o Turn off the power and clean the shaker platform to keep the equipment tidy.
6. Notes
o Do not overload the shaker or place containers that are too large;
o For cell or microbial culture, disinfect the shaker regularly to prevent contamination.
NanoDrop Spectrophotometer
Used for rapid determination of the concentration and purity of nucleic acids, proteins, or other samples.
Materials:- Samples to be measured (e.g., DNA, RNA, or protein solutions)
- Deionized water or buffer (for blank calibration)
- Lint-free tissue (e.g., Kimwipes, for wiping the measurement pedestal)
1. Turn on the instrument, launch the NanoDrop software, and select the appropriate measurement mode (e.g., Nucleic Acid, Protein, UV-Vis, etc.);
2. Open the measurement arm, pipette 1–2 µL of buffer or deionized water onto the lower pedestal, close the arm, and perform blank calibration (Blank);
3. Open the arm, wipe the upper and lower pedestals with lint-free tissue to keep them clean;
4. Pipette 1–2 µL of the sample onto the pedestal, close the arm, and click Measure to start detection;
5. Record the sample’s concentration (ng/µL or mg/mL), purity ratios (e.g., A260/A280), and other relevant data;
6. After measurement, wipe the pedestal with lint-free tissue, and optionally add deionized water again to clean;
7. After use, close the software and turn off the instrument.
Notes:- Sample volume should be limited to 1–2 µL to avoid contamination and waste;
- Clean the pedestal after each measurement to prevent residues from affecting subsequent samples;
- Regularly calibrate and maintain the instrument to ensure measurement accuracy.
Spectrophotometer
Used to measure the light absorption of samples at specific wavelengths, thereby analyzing their concentration, purity, or chemical properties.
Materials:- Sample solution to be measured
- Cuvettes (quartz or glass, depending on detection wavelength)
- Blank control solution (e.g., deionized water or buffer)
1. Turn on the instrument and allow it to preheat for a few minutes until the light source stabilizes;
2. Select and set the detection wavelength according to experimental requirements;
3. Rinse the cuvette with deionized water or buffer, dry the outer walls, and avoid fingerprints or water droplets that may affect light transmission;
4. Fill the cuvette with the blank control solution, place it into the cuvette holder, close the lid, and press the Blank button to perform blank calibration;
5. Remove the blank cuvette and replace it with the sample-filled cuvette, ensuring consistent placement orientation (transparent sides facing the light path);
6. Record the absorbance (Abs) or transmittance (T%) of the sample at the set wavelength;
7. If a standard curve is required, measure a series of standard samples with concentration gradients;
8. After measurement, clean the cuvettes and turn off the instrument.
Notes:- The outer walls of cuvettes must remain clean and transparent, free of bubbles or residual liquid;
- Quartz cuvettes must be used when detecting in the UV range (200–350 nm);
- Sample volume should be sufficient to fill the cuvette’s light path, typically 2/3–3/4 full.
Electrophoresis Apparatus and Power Supply
Used for the separation and detection of nucleic acids (DNA/RNA) or protein samples.
Materials:- Agarose or polyacrylamide gels
- Electrophoresis buffer (e.g., TAE, TBE, SDS-PAGE buffer, etc.)
- Samples to be tested and loading buffer
- Electrophoresis tank and power supply
- DNA/RNA stain or protein staining reagents
1. Gel preparation
o Prepare agarose or polyacrylamide gel solution according to experimental requirements. Add appropriate dye (e.g., EB or a safer alternative), pour into the gel mold, and insert the comb. After solidification, carefully remove the comb.
2. Sample loading preparation
o Place the solidified gel into the electrophoresis tank and add sufficient electrophoresis buffer to cover the gel surface;
o Mix samples with loading buffer and carefully pipette into the gel wells. Load a molecular weight marker (ladder) in one well.
3. Running electrophoresis
o Cover the electrophoresis tank and connect it to the power supply, ensuring correct electrode orientation (DNA/RNA migrates toward the positive electrode);
o Set appropriate voltage/current (typically 80–150 V for agarose gels; SDS-PAGE usually starts at 80–120 V, then increases to 120–180 V);
o Start the power supply to begin electrophoresis.
4. Completion and processing
o When the tracking dye has migrated to the desired position, turn off the power and disconnect the electrodes;
o Carefully remove the gel for UV visualization (nucleic acids) or staining/destaining procedures (proteins).
5. Notes
o Ensure sufficient electrophoresis buffer to prevent the gel from drying out during the run;
o Load samples gently to avoid breaking wells or causing diffusion;
o Always wear protective goggles and shields when using UV light to avoid UV damage;
o Never touch electrodes during electrophoresis. Observe proper electrical safety.
Semi-dry Transfer Apparatus
Used after protein electrophoresis (SDS-PAGE) to rapidly transfer proteins from the gel onto PVDF or NC membranes for subsequent Western Blot analysis.
Materials:- Gel after protein electrophoresis
- PVDF or NC membrane (membrane requires pretreatment, e.g., PVDF needs methanol activation)
- Filter paper (cut to the same size as the gel)
- Transfer buffer
- Semi-dry transfer apparatus and power supply
1. Membrane and gel preparation
o Immerse the PVDF membrane in methanol for 1 minute, then equilibrate it in transfer buffer;
o NC membranes can be directly equilibrated in transfer buffer;
o After electrophoresis, remove the gel, trim off excess parts, and equilibrate the gel in transfer buffer.
2. Assembly of transfer sandwich (from bottom to top)
o Filter paper (pre-soaked in transfer buffer)
o PVDF/NC membrane
o Gel
o Filter paper (pre-soaked in transfer buffer)
o Carefully remove any air bubbles using a roller to ensure even contact.
3. Transfer operation
o Place the assembled sandwich into the semi-dry transfer apparatus with the membrane on the anode side and the gel on the cathode side;
o Cover with the upper electrode plate, ensuring good contact;
o Connect the power supply and set the appropriate current/voltage (common conditions: current 0.8–1 mA/cm², transfer time 30–90 minutes, adjusted according to gel thickness and protein size);
o Start the transfer program.
4. Completion of transfer
o Turn off the power, disassemble the sandwich, and remove the membrane and gel;
o Immediately mark the membrane orientation (e.g., cut the top-left corner) and proceed with blocking and immunoblotting as required.
5. Notes
o All filter papers must be fully soaked to prevent dry spots that cause uneven transfer;
o Avoid air bubbles during assembly, as they will lead to blank spots on the membrane;
o Do not apply excessive current for long durations, as overheating can inactivate proteins.
Polymerase Chain Reaction (PCR) Thermocycler
Used for in vitro amplification of specific DNA fragments to facilitate molecular biology studies such as cloning, gene detection, and mutation analysis.
Materials:- ddH₂O
- PCR buffer
- dNTPs
- Template DNA
- Forward and reverse primers
- DNA polymerase (e.g., Taq polymerase)
- PCR tubes
1. Prepare the reaction mixture
o On ice, prepare the PCR mixture (ddH₂O, buffer, dNTPs, primers, template DNA, DNA polymerase). Mix thoroughly and dispense into PCR tubes.
2. Power on and program setup
o Turn on the PCR thermocycler;
o Set the amplification program on the control panel or software, including:
Initial denaturation (e.g., 94–95 °C, 2–5 minutes);
Cycling parameters:
Denaturation (94–95 °C, 30 seconds);
Annealing (50–65 °C, 30 seconds);
Extension (72 °C, 30 seconds–2 minutes, depending on fragment size);
Number of cycles (typically 25–40);
Final extension (72 °C, 5–10 minutes);
Hold (4 °C).
3. Load samples
o Open the lid of the thermocycler and place the PCR tubes into the sample block, ensuring tight contact with the metal module;
o Close and lock the lid.
4. Run the program
o Confirm the program settings, then start the run;
o The thermocycler will automatically perform the programmed amplification cycles.
5. Completion
o After amplification, remove the PCR tubes;
o Turn off the power and clean the workspace.
- PCR tubes must be compatible with the instrument model to avoid uneven heating;
- Always prepare the reaction mixture on ice to minimize nonspecific amplification;
- Ensure the thermocycler is properly calibrated and temperature control is accurate before use.
Estain L1 Protein Staining Instrument
Used for rapid fixation and staining of protein gels after electrophoresis, commonly applied in protein detection and analysis following SDS-PAGE.
Materials:- Protein gel after electrophoresis
- Distilled water
- Filter paper
- Gel fixing clamp (with mesh)
- Estain L1 protein staining instrument
1. Add distilled water into the tray;
2. Carefully remove the gel after electrophoresis and immerse it in the tray for about 1 minute (Note: If using Life Tech. GEL, trim off any excess protrusions);
3. Open the gel fixing clamp and place it flat on the lab bench;
4. Place the pre-soaked gel onto the mesh surface of the clamp, ensuring the gel wells face the central axis and are positioned close to it (but not beyond the mesh surface);
5. Take a piece of filter paper, soak it in the tray, and place it flat on top of the gel;
6. Close the gel fixing clamp securely;
7. Insert the gel fixing clamp into the instrument channel, ensuring the correct orientation (mesh surface facing the operator);
8. Press the START B button for the corresponding channel to begin operation. The screen will display a countdown, and the START B button will flash;
9. Before the operation ends, the instrument will emit a beep signal;
10. After completion, the instrument will continue beeping. Press the START B button again to stop, and the screen will return to the default display.
Notes:- Ensure the gel is fully soaked before operation to avoid cracking or uneven staining;
- Always check orientation when inserting the gel fixing clamp to avoid incorrect operation;
- After use, clean the tray and fixing clamp promptly to keep the instrument in good condition.
BG-gdsUVIEW Ultraviolet Analyzer
Used to observe and analyze nucleic acid electrophoresis gel results and perform gel excision, commonly applied in DNA recovery and subsequent molecular cloning experiments.
Materials:- Nucleic acid gel after electrophoresis
- Gel cutter or scalpel blade
- Sample tray
- Camera or imaging system (optional)
1. Startup preparation
o Connect the instrument to a 220V power supply and turn on the main power switch;
o Pull out the drawer-style light box and place the sample on the tray.
2. Sample observation
o Turn on the light source switch;
o Lift the protective cover of the observation window to directly view the electrophoresis bands;
o If photography is required, close the protective cover of the observation window, open the lens cap, insert the camera lens into the lens port, and align with the sample for imaging.
3. Gel excision
o Open the side observation windows;
o Hold the gel cutter with both hands and insert them into the dark chamber to operate;
o Simultaneously observe the electrophoresis bands through the observation window and cut out the target band according to the desired size.
4. Completion
o Remove the excised gel block and sample;
o Clean the inside of the dark chamber to maintain instrument cleanliness;
o Turn off the light source and the main power switch.
- Ultraviolet light is harmful to eyes and skin. Always wear protective goggles or use the observation window’s protective device during operation;
- Perform gel cutting gently to avoid damaging the target band;
- Clean the sample tray and gel cutter promptly to prevent cross-contamination;
- Disconnect the power supply and cover the instrument with a dust cover when not in use for extended periods.
Microplate Reader
Used to detect optical signals such as absorbance (OD), fluorescence, or luminescence in 96-well or 384-well microplates. Commonly applied in ELISA, enzyme activity assays, and cell-based experiments.
Materials:- Prepared 96-well or 384-well microplate
- Samples and controls required for the experiment (standards, positive/negative controls)
- Microplate reader and supporting software
1. Startup and preparation
o Turn on the microplate reader and launch the software;
o Select the appropriate detection mode (e.g., absorbance, fluorescence, luminescence) based on experimental requirements.
2. Sample preparation
o Add samples, standards, and controls into the designated wells of the microplate;
o If incubation or color development is required, ensure these steps are completed before measurement.
3. Detection settings
o Place the microplate into the reader tray, ensuring the correct orientation;
o Set detection parameters in the software, such as wavelength (commonly 450 nm, 570 nm, 600 nm, etc.), reading mode (single-point or multi-point), and plate reading sequence.
4. Running and data acquisition
o Start the detection program. The instrument will automatically read each well;
o Once reading is completed, results will be displayed in the software interface;
o Export data in Excel, PDF, or other formats for further analysis.
5. Completion
o Remove the microplate and properly dispose of waste solutions;
o Close the software and power off the instrument;
o Clean the tray and workspace to maintain instrument hygiene.
- The bottom of the microplate must be clean and free of bubbles, as these may affect accuracy;
- Select the appropriate detection mode and wavelength depending on the experiment;
- Regular wavelength calibration and optical path maintenance are required to ensure measurement accuracy.
Gel Filtration Chromatography / Size-Exclusion Chromatography (SEC, Molecular Sieve)
Used to separate biomacromolecules such as proteins, peptides, and polysaccharides based on molecular size. Common applications include sample purification, buffer exchange, and detection of protein aggregation states.
Materials:- Chromatography column (packed with size-exclusion media such as Sephadex, Superdex, or Sephacryl)
- Sample solution to be separated
- Chromatography buffer (filtered and degassed)
- Chromatography system (including pump, injection valve, detector, and fraction collector)
- Collection tubes
1. System preparation
o Check all connections in the chromatography system to ensure there are no leaks;
o Equilibrate the column with filtered and degassed buffer (typically 1–2 column volumes, CV);
o Set an appropriate flow rate (commonly 0.5–1.0 mL/min, depending on column specifications).
2. Sample preparation
o Clarify samples by centrifugation or filtration (0.22 µm) to remove particulates or precipitates;
o Sample volume should not exceed 2–5% of the column volume to maintain resolution.
3. Sample loading and separation
o Inject the sample via the sample loop or injection port, then switch to load mode;
o Start buffer pumping to initiate separation;
o Monitor UV absorbance in real time (typically at 280 nm) or other detection signals.
4. Fraction collection
o Collect fractions based on chromatogram peak profiles, labeling tube numbers accordingly;
o Further analyze fractions for purity if required (e.g., SDS-PAGE, Western blot).
5. Shutdown and storage
o After separation, wash the column with 1–2 CV of buffer;
o For long-term storage, wash the column with storage solution (e.g., 20% ethanol) and seal properly;
o Turn off the system power and clean the sample loop and tubing.
- Avoid air bubbles in the column; always degas the buffer before use;
- Never allow the column bed to dry, keep it constantly hydrated;
- Control sample concentration and injection volume to prevent overloading and loss of resolution;
- Monitor system pressure during operation to avoid column damage due to overpressure.
Optical Microscope
Used to observe the morphology and structure of cells, tissue sections, microorganisms, or other transparent/semi-transparent samples.
Materials:- Prepared specimen slides (e.g., tissue sections, cell smears)
- Cover slips
- Tweezers, dropper (as needed)
1. Preparation before use
o Place the microscope on a stable workbench and connect the power supply;
o Turn on the light source and adjust the brightness to a suitable level;
o Check whether the objective lenses and eyepieces are clean; if dusty, wipe gently with lens paper.
2. Placing the sample
o Place the specimen slide at the center of the stage and secure it with stage clips;
o Select the lowest magnification objective (usually 4× or 10×), and rotate the coarse focus knob to bring the objective close to the sample.
3. Adjustment and observation
o Observe through the eyepiece, slowly rotate the coarse focus knob backward until the sample comes into clear focus;
o Use the fine focus knob to improve sharpness;
o Switch gradually to higher magnification objectives (40×, 100× oil immersion, etc.) as needed, and use fine focus after each change.
4. After use
o Return to the low-power objective and lower the stage;
o Turn off the light source and unplug the power;
o Wipe the lenses and stage to keep the microscope clean;
o Cover with a dust-proof cover and store in a dry environment.
- When using the oil immersion lens (100×), add cedar oil and clean it immediately after use;
- Avoid touching the lenses directly with fingers;
- Do not use the coarse focus knob with high-power objectives to prevent damage to the slide or lens.
Scanning Electron Microscope (SEM)
Used to observe the microscopic morphology and structural features of sample surfaces. Commonly applied in materials science, biology, and nanotechnology research.
Materials:- Samples to be tested (solid samples or biological samples that have been fixed and dried)
- Conductive adhesive or sample holder (stub)
- Metal coating materials (e.g., gold, platinum; for sputter-coating non-conductive samples)
- SEM instrument and control software
1. Sample preparation
o Solid samples: cut to an appropriate size and mount onto the sample holder;
o Biological or non-conductive samples: fix, dehydrate, dry, and sputter-coat with metal to enhance conductivity.
2. Sample loading
o Attach the sample to the stub using conductive adhesive;
o Place the sample holder into the SEM chamber and close the chamber door.
3. Startup and settings
o Turn on the SEM power; sequentially start the vacuum system, cooling system, and electron gun high voltage;
o Wait until the chamber reaches the required vacuum level;
o Set parameters in the control software, including accelerating voltage, magnification, and detector mode (secondary electron or backscattered electron imaging).
4. Imaging and adjustment
o Activate the electron beam and move the stage to locate the target area;
o Adjust focus, brightness, contrast, and working distance until the image is clear and stable;
o Observe multiple regions and magnifications as needed, and acquire images.
5. After use
o Turn off the electron beam and high-voltage power supply;
o Sequentially shut down the vacuum pump, cooling system, and main power;
o Remove the sample and clean the sample stage.
- Samples must be dry and conductive; otherwise, charging artifacts may occur and distort the image;
- Avoid rapid switching of high voltage or frequent on/off of the electron gun to prevent damage;
- Do not focus at high magnification on the same region for extended periods to reduce electron beam damage;
- Only trained personnel should operate SEM, with familiarity in vacuum and high-voltage procedures.
Talos 200S Transmission Electron Microscope (TEM)
Used to observe ultrastructures of cells or proteins, such as nanodiscs.
Materials:- Prepared samples
- 300-mesh carbon support grids
1. Startup
o Sequentially turn on the TEM main power, vacuum pump, cooling system, and high-voltage power supply;
o Wait for the instrument to complete self-checks and then enter the operation software interface.
2. Sample loading
o Carefully mount the prepared sample onto the TEM loading rod;
o Ensure the sample is positioned accurately and securely fixed.
3. Parameter setup
o In the software interface, set appropriate parameters such as accelerating voltage, magnification, and focusing mode.
4. Imaging and analysis
o Activate the electron beam;
o Adjust the sample stage position, focusing rings, and apertures until the sample image is clearly displayed on the screen;
o Import recorded images or data into the computer for further processing and analysis.
5. Shutdown
o Turn off the electron beam and high-voltage power supply;
o Sequentially shut down the vacuum pump, cooling system, and main power supply.
- Ensure samples are properly prepared and mounted to avoid beam damage or instability;
- Always follow correct vacuum and high-voltage operating procedures;
- Only trained personnel should perform TEM operations.
Dynamic Light Scattering (DLS) Instrument
Used to measure the particle size distribution, average hydrodynamic diameter, and aggregation state of nanoparticles, proteins, or colloids. It is commonly applied to assess the homogeneity and stability of samples.
Materials:- Sample solution (proteins, nanoparticles, or colloids)
- Filtered buffer or solvent (for dilution and blank control)
- Disposable or quartz cuvettes (compatible with the DLS instrument)
- Micropipette and sterile tips
1. Sample preparation
o Centrifuge or filter the sample (e.g., through a 0.22 μm filter) to remove dust and large particles;
o Dilute the sample to an appropriate concentration to ensure scattering intensity is within the measurable range.
2. Instrument startup and settings
o Turn on the DLS instrument and launch the operating software;
o Select the desired measurement mode (particle size distribution, Zeta potential, etc.);
o Set the experimental parameters such as temperature, detection angle, and acquisition time.
3. Sample loading
o Use a pipette to transfer the sample into the cuvette, avoiding air bubbles;
o Place the cuvette into the sample holder, ensuring correct positioning.
4. Measurement process
o Click Measure to start data collection;
o The instrument analyzes fluctuations in scattered light caused by Brownian motion to calculate particle size;
o The software generates results automatically, including average hydrodynamic diameter and PDI (polydispersity index).
5. Experiment completion
o Record and export the measurement results (Excel or PDF format);
o Remove the cuvette, clean it immediately, and let it dry;
o Shut down the software and power off the instrument;
o Keep the instrument clean and dust-free.
- Samples must be clear and free of bubbles, as these can cause significant artifacts;
- The concentration should be optimal: too high leads to multiple scattering, while too low results in weak signals;
- Ensure correct input of solvent parameters such as refractive index and viscosity for accurate calculation;
- Avoid vibrations and external light interference during measurement.
pH Meter
Used to measure the acidity or alkalinity (pH value) of solutions. Widely applied in biological experiments, chemical experiments, and culture medium preparation.
Materials:- pH meter main unit
- Combination electrode or glass electrode
- Standard buffer solutions (pH 4.00, 7.00, 10.00)
- Sample solution
- Distilled or deionized water (for electrode rinsing)
- Soft lint-free tissue
1. Instrument preparation
o Turn on the power and allow the instrument to warm up for a few minutes;
o Check the electrode condition: the glass bulb should be filled with solution and not dry.
2. Calibration
o Rinse the electrode with distilled water and gently wipe dry;
o Immerse the electrode in the pH 7.00 standard buffer, press the Calibrate button;
o Follow software prompts to calibrate with pH 4.00 and pH 10.00 buffers in sequence to ensure measurement accuracy.
3. Sample measurement
o Rinse the electrode and gently dry;
o Immerse the electrode in the test solution, stir lightly, then let it stand until the reading stabilizes;
o Record the pH value.
4. End of operation
o After use, rinse the electrode with distilled water and wipe dry;
o For long-term storage, immerse the electrode in special storage solution (not dry);
o Turn off the power and keep the instrument and electrode clean.
- The electrode should not touch hard surfaces or be over-wiped to avoid damaging the glass bulb;
- Always use fresh standard buffers for calibration;
- Between different samples, the electrode must be thoroughly rinsed to avoid cross-contamination;
- For long-term storage, keep the electrode in electrode storage solution or KCl solution.
High-Performance Liquid Chromatography (HPLC)
Used for separation, identification, and quantitative analysis of compounds in samples. Widely applied in pharmaceutical testing, protein purification, metabolite analysis, and other fields.
Materials:- Prepared and filtered sample solution
- Mobile phase (filtered and degassed)
- Chromatography column (e.g., C18, ion-exchange, size-exclusion, depending on experiment)
- HPLC system (pump, injector, detector, and data acquisition system)
- Sample vials and injection syringe
1. System preparation
o Switch on the HPLC power, start the pump and detector;
o Flush the system with filtered and degassed mobile phase until the baseline stabilizes;
o Equilibrate the chromatography column (typically 10–20 minutes).
2. Sample preparation
o Filter the sample through a 0.22 µm or 0.45 µm membrane to remove particulates;
o Transfer to sample vials, place in autosampler tray or prepare for manual injection.
3. Chromatographic run
o Set detection parameters: mobile phase composition, flow rate, column temperature, detection wavelength, etc.;
o Program gradient elution if required;
o Inject the sample and start the run, recording chromatogram.
4. Data acquisition and analysis
o Use software to record signals and generate chromatograms;
o Perform quantitative analysis based on peak area or height;
o Identify compounds by comparing retention times with reference standards.
5. End of operation
o Flush the system with appropriate solvent (e.g., 100% water or methanol) to clean the column;
o For long-term storage, keep the column in the recommended storage solution;
o Turn off the pump, detector, and main power.
- The mobile phase must be filtered and degassed to prevent bubbles interfering with detection;
- Samples must be clear and particle-free to avoid clogging the column;
- System pressure should remain within the recommended operating range of the column to prevent damage;
- Regularly clean the injector, sample loop, and flow path to maintain system performance.
Reagent Formulation
Culture Media
LB Medium (Luria-Bertani Medium)
Formula:
- Tryptone 0.01 g/mL (10 g/L)
- Yeast extract 0.005 g/mL (5 g/L)
- Sodium chloride 0.01 g/mL (10 g/L)
- Deionized water
OR - Pre-mixed LB powder 0.025 g/mL (25 g/L)
- Deionized water
- 100 mL
Tryptone 1 g, Yeast extract 0.5 g, NaCl 1 g, Deionized water to 100 mL OR Pre-mixed LB powder 2.5 g, Deionized water to 100 mL - 200 mL
Tryptone 2 g, Yeast extract 1 g, NaCl 2 g, Deionized water to 200 mL OR Pre-mixed LB powder 5 g, Deionized water to 200 mL - 500 mL
Tryptone 5 g, Yeast extract 2.5 g, NaCl 5 g, Deionized water to 500 mL OR Pre-mixed LB powder 12.5 g, Deionized water to 500 mL - 1 L
Tryptone 10 g, Yeast extract 5 g, NaCl 10 g, Deionized water to 1 L OR Pre-mixed LB powder 25 g, Deionized water to 1 L - 2 L
Tryptone 20 g, Yeast extract 10 g, NaCl 20 g, Deionized water to 2 L OR Pre-mixed LB powder 50 g, Deionized water to 2 L - 3 L
Tryptone 30 g, Yeast extract 15 g, NaCl 30 g, Deionized water to 3 L OR Pre-mixed LB powder 75 g, Deionized water to 3 L - 4 L
Tryptone 40 g, Yeast extract 20 g, NaCl 40 g, Deionized water to 4 L OR Pre-mixed LB powder 100 g, Deionized water to 4 L - 5 L
Tryptone 50 g, Yeast extract 25 g, NaCl 50 g, Deionized water to 5 L OR Pre-mixed LB powder 125 g, Deionized water to 5 L
Autoclave at 121 °C for 20 min.
Storage:Room temperature.
Notes:1.Agar is not added when preparing LB broth.
2.Antibiotics are not added before autoclaving, since they are heat-labile. Add the appropriate antibiotic only after sterilization and cooling, depending on experimental requirements.
LB Semi-Solid Medium Formula:- Tryptone 0.01 g/mL (10 g/L)
- Yeast extract 0.005 g/mL (5 g/L)
- Sodium chloride 0.01 g/mL (10 g/L)
- Agar powder 0.3%–0.7%
- Deionized water
OR - Pre-mixed LB powder 0.025 g/mL (25 g/L)
- Agar powder 0.3%–0.7%
- Deionized water
- 100 mL
Tryptone 1 g, Yeast extract 0.5 g, NaCl 1 g, Agar 0.3–0.7 g, Deionized water to 100 mL OR Pre-mixed LB powder 2.5 g, Agar 0.3–0.7 g, Deionized water to 100 mL - 200 mL
Tryptone 2 g, Yeast extract 1 g, NaCl 2 g, Agar 0.6–1.4 g, Deionized water to 200 mL OR Pre-mixed LB powder 5 g, Agar 0.6–1.4 g, Deionized water to 200 mL - 500 mL
Tryptone 5 g, Yeast extract 2.5 g, NaCl 5 g, Agar 1.5–3.5 g, Deionized water to 500 mL OR Pre-mixed LB powder 12.5 g, Agar 1.5–3.5 g, Deionized water to 500 mL - 1 L
Tryptone 10 g, Yeast extract 5 g, NaCl 10 g, Agar 3–7 g, Deionized water to 1 L OR Pre-mixed LB powder 25 g, Agar 3–7 g, Deionized water to 1 L - 2 L
Tryptone 20 g, Yeast extract 10 g, NaCl 20 g, Agar 6–14 g, Deionized water to 2 L OR Pre-mixed LB powder 50 g, Agar 6–14 g, Deionized water to 2 L - 3 L
Tryptone 30 g, Yeast extract 15 g, NaCl 30 g, Agar 9–21 g, Deionized water to 3 L OR Pre-mixed LB powder 75 g, Agar 9–21 g, Deionized water to 3 L - 4 L
Tryptone 40 g, Yeast extract 20 g, NaCl 40 g, Agar 12–28 g, Deionized water to 4 L OR Pre-mixed LB powder 100 g, Agar 12–28 g, Deionized water to 4 L - 5 L
Tryptone 50 g, Yeast extract 25 g, NaCl 50 g, Agar 15–35 g, Deionized water to 5 L OR Pre-mixed LB powder 125 g, Agar 15–35 g, Deionized water to 5
Autoclave at 121 °C for 20 minutes.
Storage:Room temperature.
Notes:1.Agar must be added when preparing LB semi-solid medium, and it should be sterilized by autoclaving.
2.Do not add antibiotics before autoclaving, as they are heat-labile. Add antibiotics after sterilization and cooling, according to experimental requirements.
LB Solid Medium Formula:- Tryptone 0.01 g/mL (10 g/L)
- Yeast extract 0.005 g/mL (5 g/L)
- Sodium chloride 0.01 g/mL (10 g/L)
- Agar powder 1.5%–2%
- Deionized water
OR - Pre-mixed LB powder 0.025 g/mL (25 g/L)
- Agar powder 1.5%–2%
- Deionized water
- 100 mL
Tryptone 1 g, Yeast extract 0.5 g, NaCl 1 g, Agar 1.5–2 g, Deionized water to 100 mL OR Pre-mixed LB powder 2.5 g, Agar 1.5–2 g, Deionized water to 100 mL - 200 mL
Tryptone 2 g, Yeast extract 1 g, NaCl 2 g, Agar 3–4 g, Deionized water to 200 mL OR Pre-mixed LB powder 5 g, Agar 3–4 g, Deionized water to 200 mL - 500 mL
Tryptone 5 g, Yeast extract 2.5 g, NaCl 5 g, Agar 7.5–10 g, Deionized water to 500 mL OR Pre-mixed LB powder 12.5 g, Agar 7.5–10 g, Deionized water to 500 mL - 1 L
Tryptone 10 g, Yeast extract 5 g, NaCl 10 g, Agar 15–20 g, Deionized water to 1 L OR Pre-mixed LB powder 25 g, Agar 15–20 g, Deionized water to 1 L - 2 L
Tryptone 20 g, Yeast extract 10 g, NaCl 20 g, Agar 30–40 g, Deionized water to 2 L OR Pre-mixed LB powder 50 g, Agar 30–40 g, Deionized water to 2 L - 3 L
Tryptone 30 g, Yeast extract 15 g, NaCl 30 g, Agar 45–60 g, Deionized water to 3 L OR Pre-mixed LB powder 75 g, Agar 45–60 g, Deionized water to 3 L - 4 L
Tryptone 40 g, Yeast extract 20 g, NaCl 40 g, Agar 60–80 g, Deionized water to 4 L OR Pre-mixed LB powder 100 g, Agar 60–80 g, Deionized water to 4 L - 5 L
Tryptone 50 g, Yeast extract 25 g, NaCl 50 g, Agar 75–100 g, Deionized water to 5 L OR Pre-mixed LB powder 125 g, Agar 75–100 g, Deionized water to 5 L
Sterilization:
Autoclave at 121 °C for 20 minutes.
Storage:Room temperature.
Notes:1.Agar must be added when preparing LB solid medium, and it should be sterilized by autoclaving.
2.Do not add antibiotics before autoclaving, as they are heat-labile. Add antibiotics after sterilization and cooling, according to experimental requirements.
3.If LB solid medium is to be poured into plates or dispensed into tubes, re-melt the medium using a microwave or heating block after sterilization. Once fully liquefied, pour into Petri dishes or tubes under aseptic conditions.
M9 Minimal Medium (Liquid)
- KH₂PO₄ 0.003 g/mL (0.3 g/L)
- Na₂HPO₄ 0.00678 g/mL (6.78 g/L) or Na₂HPO₄·12H₂O 0.0171 g/mL (17.1 g/L)
- NaCl 0.0005 g/mL (0.5 g/L)
- NH₄Cl 0.001 g/mL (1 g/L)
- MgSO₄ 0.000241 g/mL (0.241 g/L) or MgSO₄·7H₂O 0.000493 g/mL (0.493 g/L)
- CaCl₂ 0.000011 g/mL (0.011 g/L) or CaCl₂·2H₂O 0.0000147 g/mL (0.0147 g/L)
- Glucose 0.004 g/mL (4 g/L)
- Deionized water
- 100 mL
KH₂PO₄ 0.03 g, Na₂HPO₄ 0.678 g or Na₂HPO₄·12H₂O 1.71 g, NaCl 0.05 g, NH₄Cl 0.1 g, MgSO₄ 0.0241 g or MgSO₄·7H₂O 0.0493 g, CaCl₂ 0.0011 g or CaCl₂·2H₂O 0.00147 g, Glucose 0.4 g, dH₂O to 100 mL - 200 mL
KH₂PO₄ 0.06 g, Na₂HPO₄ 1.356 g or Na₂HPO₄·12H₂O 3.42 g, NaCl 0.1 g, NH₄Cl 0.2 g, MgSO₄ 0.0482 g or MgSO₄·7H₂O 0.0986 g, CaCl₂ 0.0022 g or CaCl₂·2H₂O 0.00294 g, Glucose 0.8 g, dH₂O to 200 mL - 500 mL
KH₂PO₄ 0.15 g, Na₂HPO₄ 3.39 g or Na₂HPO₄·12H₂O 8.55 g, NaCl 0.25 g, NH₄Cl 0.5 g, MgSO₄ 0.1205 g or MgSO₄·7H₂O 0.2465 g, CaCl₂ 0.0055 g or CaCl₂·2H₂O 0.00735 g, Glucose 2 g, dH₂O to 500 mL - 1 L
KH₂PO₄ 0.3 g, Na₂HPO₄ 6.78 g or Na₂HPO₄·12H₂O 17.1 g, NaCl 0.5 g, NH₄Cl 1 g, MgSO₄ 0.241 g or MgSO₄·7H₂O 0.493 g, CaCl₂ 0.011 g or CaCl₂·2H₂O 0.0147 g, Glucose 4 g, dH₂O to 1 L - 2 L
KH₂PO₄ 0.6 g, Na₂HPO₄ 13.56 g or Na₂HPO₄·12H₂O 34.2 g, NaCl 1 g, NH₄Cl 2 g, MgSO₄ 0.482 g or MgSO₄·7H₂O 0.986 g, CaCl₂ 0.022 g or CaCl₂·2H₂O 0.0294 g, Glucose 8 g, dH₂O to 2 L - 5 L
KH₂PO₄ 1.5 g, Na₂HPO₄ 33.9 g or Na₂HPO₄·12H₂O 85.5 g, NaCl 2.5 g, NH₄Cl 5 g, MgSO₄ 1.205 g or MgSO₄·7H₂O 2.465 g, CaCl₂ 0.055 g or CaCl₂·2H₂O 0.0735 g, Glucose 20 g, dH₂O to 5 L
- Ideally, autoclave all components except glucose at 121 °C for 20 minutes.
- Prepare glucose separately as a sterile stock solution (filter sterilized) and add aseptically after autoclaving.
- To simplify, M9 medium can also be autoclaved at 115 °C for 20 minutes, but never at 121 °C with glucose due to caramelization.
Room temperature.
Notes:1.M9 minimal medium does not contain agar (for liquid culture).
2.Antibiotics should not be added before sterilization (they are heat-labile). Add them after autoclaving, as required.
3.Glucose must not be autoclaved at 121 °C; it caramelizes and becomes unusable. Always sterilize separately by filtration.
4.Due to the very small amounts of MgSO₄ and CaCl₂ required, prepare concentrated sterile stock solutions for accuracy and add by volume. These can be sterilized separately and mixed with the base medium under aseptic conditions.
5.Only prepare 1× M9. Do not prepare concentrated stock solutions for later dilution.
Antibiotics
Ampicillin (Amp)
50 μg/mL
Stock concentration:50 μg/mL
Addition ratio:1:1000
Common preparation volumes:- 5 mL
250 mg Ampicillin, add dH₂O to 5 mL - 10 mL
500 mg Ampicillin, add dH₂O to 10 mL - 15 mL
750 mg Ampicillin, add dH₂O to 15 mL - 20 mL
1000 mg Ampicillin, add dH₂O to 20 mL
Filter sterilization (0.22 μm membrane)
Storage:4 °C, protected from light
Notes:1.Antibiotics are heat-labile and must not be autoclaved; filter sterilization is required.
2.Do not add antibiotics to freshly autoclaved media while still hot. Always wait until the medium has cooled sufficiently (≈50 °C) before adding. Ampicillin is light-sensitive and degrades under UV exposure; avoid exposing solutions to UV lamps in laminar flow hoods.
Kanamycin (Kan)
50 μg/mL
Stock concentration:50 mg/mL
Addition ratio:1:1000
Common preparation volumes:- 5 mL
250 mg Kanamycin, add dH₂O to 5 mL - 10 mL
500 mg Kanamycin, add dH₂O to 10 mL - 15 mL
750 mg Kanamycin, add dH₂O to 15 mL - 20 mL
1000 mg Kanamycin, add dH₂O to 20 mL
Filter sterilization (0.22 μm membrane)
Storage:4 °C, protected from light
Note:1.Kanamycin is heat-sensitive and must not be autoclaved; sterilization should be performed by filtration.
2.Do not add Kanamycin into freshly autoclaved media while still hot. Allow the medium to cool adequately (~50 °C) before addition.
3.Kanamycin is degraded by UV light; avoid exposing solutions to UV in laminar flow hoods.
Chloramphenicol (Cm)
25 μg/mL
Stock concentration:5 mg/mL
Addition ratio:1:200
Common preparation volumes:- 5 mL
25 mg Chloramphenicol, add dH₂O to 5 mL - 10 mL
50 mg Chloramphenicol, add dH₂O to 10 mL - 15 mL
75 mg Chloramphenicol, add dH₂O to 15 mL - 20 mL
100 mg Chloramphenicol, add dH₂O to 20 mL
Filter sterilization (0.22 μm membrane)
Storage:4 °C, protected from light
Notes:1.Chloramphenicol is heat-sensitive and must not be autoclaved; sterilization should be performed by filtration.
2.Do not add Chloramphenicol into freshly autoclaved media while still hot. Allow the medium to cool adequately (~50 °C) before addition.
3.Chloramphenicol is degraded by UV light; avoid exposing solutions to UV in laminar flow hoods.
Gentamicin (Gm)
40 μg/mL
Stock concentration:40 mg/mL
Addition ratio:1:1000
Common preparation volumes:- 5 mL
200 mg Gentamicin, add dH₂O to 5 mL - 10 mL
400 mg Gentamicin, add dH₂O to 10 mL - 15 mL
600 mg Gentamicin, add dH₂O to 15 mL - 20 mL
800 mg Gentamicin, add dH₂O to 20 mL
Filter sterilization (0.22 μm membrane)
Storage:4 °C, protected from light
Notes:1.Gentamicin is heat-sensitive and must not be autoclaved; sterilization should be performed by filtration.
2.Do not add Gentamicin to freshly autoclaved media while still hot. Allow the medium to cool (~50 °C) before addition.
3.Gentamicin is degraded by UV light; avoid exposing solutions to UV in laminar flow hoods.
Agarose Gel and Related Reagents
Agarose Gel
- 1.0% agarose gel (100 mL total volume)
- 1.0 g agarose powder
- 100 mL 1× TAE buffer
- 10 μL 10,000× GelStain
Not required
Storage:Prepare fresh for use; store solidified gels at 4 °C, wrapped in plastic wrap or sealed bag, up to several days.
Running Buffer (TAE buffer)
- 2 mol/L Tris-acetate
- 50 mmol/L EDTA
Dilute to 1× before use
Sterilization:Not required
Storage:4 °C
Loading Buffer
Typically glycerol, tracking dye (e.g., bromophenol blue, xylene cyanol) in suitable buffer (commercially available or lab-prepared)
Working concentration:5× or 6×, diluted to 1× in sample
Sterilization:Not required
Storage:4 °C
DNA Ladder/Marker (DNA molecular weight standard)
Mixture of DNA fragments of defined lengths, supplied by manufacturer
Usage:Typically 5–10 μL per lane, depending on ladder concentration
Storage:4 °C for short-term, –20 °C for long-term
Polyacrylamide Gels and Related Reagents
SDS-PAGE Gel (Sodium Dodecyl Sulfate–Polyacrylamide Gel)
Acrylamide (Acr), Methylene Bisacrylamide (Bis), SDS, Ammonium Persulfate (APS), TEMED, buffer, dH₂O
Typical preparation:- Separating gel (7 mL per gel, concentration depending on protein size range)
- Stacking gel (3 mL per gel)
- Example (10 mL separating gel, 10%): 4.17 mL H₂O, 3.33 mL 30%
Acr-Bis (29:1), 2.5 mL 4× Separating Buffer, 100 μL 10% APS, 4 μL TEMED - Example (4 mL stacking gel): 2.28 mL H₂O, 0.68 mL 30% Acr-Bis (29:1), 1 mL 4× Stacking Buffer, 40 μL 10% APS, 4 μL TEMED
Not required (freshly prepared before use)
Storage:- APS at –20 °C
- Other reagents at 4 °C
- Prepared gels stored in dH₂O at 4 °C (short-term use)
Native Polyacrylamide Gel
Acrylamide (Acr), Methylene Bisacrylamide (Bis), APS, TEMED, buffer, dH₂O
Sterilization:Not required
Storage:APS at –20 °C, other reagents at 4 °C; prepared gels stored in dH₂O at 4 °C
Running Buffer (SDS-PAGE Running Buffer)
3.03 g Tris, 17.99 g Glycine, 1 g SDS, add dH₂O to 1 L
Sterilization:Not required
Storage:Room temperature
Loading Buffer (Sample Buffer)
Typically contains Tris-HCl, glycerol, SDS, DTT/β-mercaptoethanol, and tracking dyes (bromophenol blue, xylene cyanol); commercially available or lab-prepared
Working concentration:2×, 5×, or 6×; dilute to 1× in protein sample before loading
Sterilization:Not required
Storage:–20 °C
Protein Ladder/Marker (Molecular Weight Standard)
Mixture of proteins of defined molecular weights, supplied by manufacturer (prestained or unstained)
Usage:Typically 5–10 μL per lane, depending on concentration
Storage:–20 °C
Protein Induction, Purification, and Quantification Reagents
IPTG (Isopropyl β-D-1-thiogalactopyranoside)
200 mg/mL in dH₂O
Working concentration (typical):0.05–1.0 mM (added to bacterial culture for protein induction)
Typical additions (for 200 mg/mL stock):- 0.05 mM: 6–24 μL per 100–400 mL culture
- 0.1 mM: 12–48 μL per 100–400 mL culture
- 0.2 mM: 24–96 μL per 100–400 mL culture
- 0.4 mM: 48–191 μL per 100–400 mL culture
- 1.0 mM: 119–477 μL per 100–400 mL culture
Filter sterilization (0.22 μm membrane)
Storage:4 °C, protected from light
Notes:Heat- and UV-sensitive; do not autoclave.
Buffer A (Lysis/Binding buffer)
50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5% glycerol, 2 mM β-mercaptoethanol, dH₂O
Sterilization:Filter sterilization recommended
Storage:4 °C
Buffer B (Binding buffer, low imidazole)
50 mM Tris-HCl (pH 8.0), 20 mM imidazole, 400 mM NaCl, 5% glycerol, 2 mM β-mercaptoethanol, dH₂O
Sterilization:Filter sterilization recommended
Storage:4 °C
Buffer C1 (Wash buffer, medium imidazole)
50 mM Tris-HCl (pH 8.0), 100 mM imidazole, 400 mM NaCl, 5% glycerol, 2 mM β-mercaptoethanol, dH₂O
Sterilization:Filter sterilization recommended
Storage:4 °C
Buffer C2 (Wash buffer, high imidazole)
50 mM Tris-HCl (pH 8.0), 300 mM imidazole, 400 mM NaCl, 5% glycerol, 2 mM β-mercaptoethanol, dH₂O
Sterilization:Filter sterilization recommended
Storage:4 °C
Buffer C3 (Elution buffer, very high imidazole)
50 mM Tris-HCl (pH 8.0), 500 mM imidazole, 400 mM NaCl, 5% glycerol, 2 mM β-mercaptoethanol, dH₂O
Sterilization:Filter sterilization recommended
Storage:4 °C
Bradford Reagent (Protein quantification dye)
Coomassie Brilliant Blue G-250 dye in acidic solution (commercial kit, 5× concentrate)
Working solution:Dilute 5× stock to 1× with dH₂O before use
Sterilization:Not required
Storage:4 °C
Dialysis Buffers
Inclusion Body Refolding Dialysis Buffer
- 4 mM GSH (reduced glutathione)
- 0.4 mM GSSG (oxidized glutathione)
- 50 mM Tris-HCl
- 25 mM NaCl
- 2 mM EDTA
- 3 mM β-mercaptoethanol
- 15% glycerol
Filter sterilization (0.22 μm membrane) recommended
Storage:4 °C
Assembly Dialysis Buffer
- 50 mM Tris-HCl
- 25 mM NaCl
- 2 mM EDTA
- 3 mM β-mercaptoethanol
- 10% glycerol
Filter sterilization (0.22 μm membrane) recommended
Storage:4 °C
Enzyme Activity Assay Reagents
Substrate Solution
- 6 mM PolyP
- 10 mM ADP
- 20 mM MgCl₂
- 100 mM Tris-HCl (pH 8.0)
Filter sterilization (0.22 μm membrane) recommended
Storage:4 °C
ATP Detection Solution
ATP assay reagent : Dilution buffer = 1 : 9
Sterilization:Not required
Storage:Prepare fresh before use; short-term storage at 4 °C possible
ATP Detection Solution
1 M KH₂PO₃ / K₂HPO₃
Sterilization:Filter sterilization (0.22 μm membrane) recommended
Storage:4 °C
Pull-down Buffers
Binding Buffer
- 50 mM Tris-HCl (pH 8.0)
- 150 mM NaCl
- 0.1% Triton X-100
- dH₂O
- 250 μL Tris-HCl (pH 8.0)
- 0.088 g NaCl
- 10 μL Triton X-100
- Add dH₂O to 10 mL final volume
Not required
Storage:Room temperature
Notes:- pH adjustment is not necessary since Tris-HCl stock is already pH 8.0.
- Due to the high viscosity of Triton X-100, use reverse pipetting or immerse the pipette tip directly into the reagent to improve accuracy.
Elution Buffer
- 0.1 M Glycine
- 0.1% Triton X-100
- dH₂O
- Adjust pH to 2.5–3.1 with HCl
- 0.075 g Glycine
- 10 μL Triton X-100
- Add dH₂O to 10 mL final volume
- Adjust pH to 2.5–3.1 with HCl
Not required
Storage:Room temperature
Notes:- Accurate pH adjustment (2.5–3.1) is critical for efficient protein elution; calibrate pH meter carefully before use.
- Triton X-100 is viscous; use reverse pipetting or immerse tip directly for accurate measurement.
Western Blot & Dot Blot Reagents
Transfer Buffer
- 25 mM Tris
- 192 mM Glycine
- 20% (v/v) Methanol
- dH₂O
- 3.03 g Tris
- 14.4 g Glycine
- 200 mL Methanol
- Add dH₂O to 1 L final volume
Not required
Storage:Room temperature
Notes:Methanol is volatile, toxic, and has a pungent odor; avoid inhalation and ensure good ventilation.
TBST Buffer
- 20 mM Tris-HCl (pH 7.4–7.6)
- 150 mM NaCl
- 0.1% (v/v) Tween-20
- dH₂O
- 10 mL of 2 M Tris-HCl stock (pH 7.4–7.6)
- 8.77 g NaCl
- 1 mL Tween-20
- Add dH₂O to 1 L final volume and adjust pH if necessary
Not required
Storage:Room temperature
Notes:- Tween-20 is highly viscous; use reverse pipetting or immerse pipette tip directly into the reagent.
- Store Tween-20 properly; avoid contamination when retrieving.
Blocking Solution
5% (w/v) Skim milk powder in TBST (alternative: BSA in TBST)
Typical preparation (1 L): 50 g Skim milk powder Dissolve in 1 L TBST oSterilization:Not required
Storage:Room temperature
Notes:Skim milk is strictly for laboratory use; not suitable for consumption.
Primary Antibody Solution
Dilute antibody to appropriate working concentration in blocking solution (skim milk/TBST or BSA/TBST, depending on protocol).
Storage:4 °C (short-term, in-use)
Notes:Antibodies are expensive and sensitive; use minimal volumes and store promptly.
Secondary Antibody Solution
Dilute antibody to appropriate working concentration in blocking solution.
Storage:4 °C (short-term, in-use)
Notes:Handle carefully; store properly to avoid degradation.
CL Detection Reagent
Mix equal volumes of Solution A (contains luminol) and Solution B (contains hydrogen peroxide) immediately before use.
Sterilization:Not required
Storage:Not storable once mixed; prepare fresh each time
Notes:Always prepare freshly; do not store mixed ECL solution as it rapidly loses activity.
Silver Staining Reagents
Fixing Solution
- 30% (v/v) Ethanol
- 12% (v/v) Acetic acid
- 0.018% (v/v) 37% Formaldehyde
- dH₂O
- 30 mL Ethanol
- 12 mL Acetic acid
- 50 μL 37% Formaldehyde
- Add dH₂O to 100 mL final volume
Not required
Storage:Room temperature
Silver Nitrate Staining Solution
- 0.02% (w/v) AgNO₃
- 0.028% (v/v) 37% Formaldehyde
- dH₂O
- 20 mg AgNO₃
- 75 μL 37% Formaldehyde
- Add dH₂O to 100 mL final volume
Not required
Storage:Prepare fresh; do not store
Developing Solution
- 6% (w/v) Sodium carbonate (Na₂CO₃)
- 0.0004% (w/v) Sodium thiosulfate (Na₂S₂O₃)
- 0.028% (v/v) 37% Formaldehyde
- (Optional: add 2 mL Sensitizing solution per 100 mL)
- dH₂O
- 6 g Sodium carbonate
- 0.4 mg Sodium thiosulfate
- 75 μL 37% Formaldehyde
- (Optional: add 2 mL Sensitizing solution)
- Add dH₂O to 100 mL final volume
Not required
Storage:Prepare fresh; do not store
Stopping (Quenching) Solution
- 1% (v/v) Acetic acid
- dH₂O
- 1 mL Acetic acid
- 99 mL dH₂O
Not required
Storage:Room temperature
Sensitizing Solution
- 0.02% (w/v) Sodium thiosulfate (Na₂S₂O₃)
- dH₂O
- 20 mg Sodium thiosulfate
- Add dH₂O to 100 mL final volume
Not required
Storage:Prepare fresh; do not store
Sucrose Gradient Solutions
Composition
Buffer A (pre-prepared)
Sucrose (analytical grade)
Typical gradient preparations (w/v):
20 g sucrose dissolved in 100 mL Buffer A
35% Sucrose Solution:35 g sucrose dissolved in 100 mL Buffer A
45% Sucrose Solution:45 g sucrose dissolved in 100 mL Buffer A
50% Sucrose Solution:50 g sucrose dissolved in 100 mL Buffer A
60% Sucrose Solution:60 g sucrose dissolved in 100 mL Buffer A
Sterilization
Filter sterilization (0.22 μm membrane) recommended if solutions are used for sensitive protein or cell experiments.
Storage
4 °C, protected from microbial contamination. Solutions are stable for 1–2 weeks.
Major Reagents and Kits Sources
Major Reagents Sources
Ni-NTA Beads 6FF (Nickel resin): LabLead
β-Mercaptoethanol: Macklin
Imidazole: Sangon Biotech
Guanidine Hydrochloride(GuHCl): Sangon Biotech
Aspartic Acid: Sangon Biotech
Sodium Hexametaphosphate: Sangon Biotech
E. coli BW25113 strain: Sangon Biotech
ArcticExpress strain: Beyotime
ADP: Mei5bio
Gentamicin Sulfate: Mei5bio
Kanamycin Sulfate: Mei5bio
Immunoprecipitation Magnetic Beads: Mei5bio
FLAG Monoclonal Antibody: Mei5bio
Neutral Protease: Mei5bio
Cysteine: Mei5bio
CMP (Cytidine Monophosphate): Mei5bio
Cytidine: Mei5bio
L-Arabinose: Mei5bio
Major Kits Sources
Native-PAGE Kit: Sangon Biotech
Glutathione ELISA Kit: Sangon Biotech
ATP Assay Kit: Beyotime
SDS-PAGE Kit: Mei5bio
BCA Protein Quantification Kit: Mei5bio
Reduced Glutathione Assay Kit: Mei5bio
Acknowledgments
We thank Sangon Biotech for sequencing services and oligonucleotide synthesis. We also acknowledge the College of Chemistry and the College of Physics and Astronomy, Beijing Normal University, the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Servicebio company for providing transmission electron microscopy (TEM) services.
Complete List of References
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