Experiments

Growth media

In this protocol, you can find how to prepare LB agar plates supplemented with ampicillin for bacterial growth and selection.

Materials:

  • LB Broth (25g/L)
  • Agar (1.5%)
  • Ampicillin (100 mg/mL stock solution; 150μg/mL working concentration)

Protocols:

  1. Prepare 150 mL LB Broth with 1.5% agar.
  2. Cool the medium to ~45 °C.
  3. Add 225 µL Ampicillin (100 mg/mL stock) to reach a working concentration of 150 µg/mL.
  4. Pour plates and store at 4 °C.

In this protocol, you can find how to prepare DF-12 complete medium supplemented with FBS and additives for routine mammalian cell culture.

Materials:

  • DF-12 (DMEM/F-12) basal medium
  • Fetal Bovine Serum (FBS) 10%
  • Sodium Pyruvate
  • L-Glutamine
  • PSA (Penicillin–Streptomycin–Amphotericin B)

*Notes:

Cells were routinely maintained in complete DF-12 medium. Prior to protein analysis (e.g., SDS-PAGE/Western blot), cultures were washed twice with PBS to remove residual serum proteins.

In this protocol, you can find how to pick single colonies and expand them into liquid LB medium for plasmid propagation.

Materials:

  • LB agar plates with single colonies (Ampicillin 150 µg/mL)
  • LB broth (25g/L)
  • Ampicillin (100 mg/mL stock solution; 150 µg/mL working concentration)
  • 1.5 mL microcentrifuge tubes (Eppendorf)
  • 4 mL culture tubes / vials
  • Shaking incubator (37 °C, ~200 rpm)

Protocols:

  1. Pick a single colony from a selective LB agar plate.
  2. Inoculate into 50µL LB broth with ampicillin (150 µg/mL) in a microcentrifuge tube.
  3. Incubate at 37 °C with shaking for 3–6 h.
  4. Transfer the starter culture into 4 mL LB broth with ampicillin (150 µg/mL).
  5. Continue shaking incubation until sufficient growth is reached.

In this protocol, you can find how to subculture adherent mammalian cells (e.g., HEK293T, Caco-2) while maintaining viability for downstream applications.

Materials:

  • Complete growth medium ( HEK293T: DF-12 + 10% FBS, see DF-12 complete page; Caco-2: DMEM)
  • PBS (Ca²⁺/Mg²⁺-free)
  • Trypsin-EDTA solution
  • 15 mL sterile conical tubes
  • cell counter
  • Trypan Blue solution (0.4%)
  • Pipettes, serological pipettes, centrifuge

Protocols:

  1. Aspirate culture medium from the cells.
  2. Wash cells once with 2 mL PBS, then aspirate completely.
  3. Add 350–400 µL trypsin-EDTA to cover the cell layer.
  4. Incubate at 37 °C for 1–2 min until cells round up and detach.
  5. Confirm under microscope that cells are separated into single cells (no sharp edges); gently tap flask if needed.
  6. Add 800–1000 µL complete medium (DF-12 + 2% FBS) to neutralize trypsin.
  7. Transfer the suspension to a 15 mL conical tube.
  8. Centrifuge at 300 × g for 3–5 min.
  9. Aspirate and discard supernatant.
  10. Flick the pellet and resuspend in fresh complete medium.
  11. Mix 30 µL Trypan Blue with 10 µL cell suspension and load onto a hemocytometer.
  12. Count viable cells and calculate seeding density.
  13. Seed cells into new culture vessels with appropriate volume of fresh medium.
  14. Return to incubator (37 °C, 5% CO₂) and culture under standard conditions.

Plasmid Extraction

In this protocol, you can find how to isolate plasmid DNA from transformed E. coli DH5α cultures using a silica column-based miniprep kit.

Materials:

  • Overnight culture of transformed E. coli (grown in LB + antibiotic)
  • Plasmid Miniprep Kit (silica column based)
  • Centrifuge (≥12,000 × g)
  • Microcentrifuge tubes (1.5 mL)
  • Nuclease-free water or TE buffer (for elution)

Protocols:

  1. Centrifuge the culture tube at 6000 rpm, 4°C for 15 min, then discard the supernatant.
  2. Reaction components (depending on culture volume):
    LB media RB LB NB Our experiment
    ≤5mL 250µL 250µL 350µL ✓
    5-10mL 500µL 500µL 700µL
    10-15mL 750µL 750µL 1050µL
    15-20mL 1000µL 1000µL 1400µL
  3. Add appropriate volume of RB (premixed with RNase A) to the cell pellet and resuspend it completely by pipetting.
  4. Add appropriate volume of LB (Blue), immediately mix by inverting the tube 4-6 times (the color of the lysate should change from opaque to bright blue).
  5. Add appropriate volume of NB (Yellow), mix by inverting the tube 5-6 times. (The color of the lysate will turn into yellow when the neutralization is complete and a yellowish precipitate will form). Incubate the lysate at room temperature for 2 minutes.
  6. Centrifuge at 12,000 × g for 5 minutes, gently transfer the supernatant to a spin column. Centrifuge at 12,000 × g for 1 minute and discard the flow-through (for supernatant more than 800µL, repeat the process once).
  7. Add 250µL TB, incubate at room temperature for 10 minutes. Centrifuge at 12,000 × g for 1 minute and discard the flow-through.
  8. Add 650µL of WB (check to make sure that ethanol has been added) to the column, Centrifuge at 12,000 × g for 1 minute. Discard the flow through.
  9. Centrifuge the column at 12,000 × g for 1-2 minutes to remove residual WB completely.
  10. Place the spin column in a clean microcentrifuge tube, add 30-50µL of Elution Buffer or sterile, distilled water directly to the center of the column matrix for higher yield, prewarm EB or ddH₂O at 60-70°C.
  11. 10. Centrifuge the column at 10,000 × g for 1 minute to elute DNA. Isolated plasmid DNA is ready to use or can be stored at -20°C.

*Notes:

The following protocol was referenced from the AllPure Plasmid MiniPrep Kit manual provided by the manufacturer.

Transformation

This protocol describes how to transform competent E. coli cells by introducing plasmid DNA, followed by selection on antibiotic-containing agar plates.

Materials:

  • Sterile water
  • Plasmid DNA (stock solution, or dried DNA resuspended in Tris-EDTA buffer; e.g., 2 µg dried DNA in 20 µL TE)
  • Competent E. coli DH5α cells (25 µL per reaction)
  • LB broth (without antibiotics)
  • LB agar plates with 150 µg/mL ampicillin
  • 42 °C water bath
  • Ice bucket
  • Sterile spreader or triangular glass rod
  • Bunsen burner (for sterilization)

Protocols:

  1. Dilute plasmid DNA to 100 ng/µL.
  2. Add plasmid to thawed competent E. coli DH5α cells and incubate on ice for 20–30 min.
  3. Heat shock at 42 °C for 1 min, then place on ice for 2 min.
  4. Add 500 µL LB and recover at 37 °C, 180 rpm for 50 min.
  5. Plate 50 µL on LB-Amp agar plate.
  6. Incubate overnight at 37 °C to obtain colonies.

Transfection

In this protocol, you can find how to transfect plasmid DNA into HEK293T cells using NTR II transfection reagent.

Materials:

  • HEK293T cells (about 80% confluency)
  • Plasmid DNA (endotoxin-free)
  • NTR II transfection reagent
  • Sterile microcentrifuge tubes
  • Shaking centrifuge (brief spins)

Protocols:

  1. Prepare plasmid DNA solution: dilute 5µg plasmid (~10µL) in 180 µL DF-12 + 2% FBS.
  2. Add 10 µL NTR II reagent to each DNA/DF-12 + 2% FBS mixture.
  3. Gently mix by pipetting or inversion, briefly spin down.
  4. Incubate the mixture at room temperature for 25–30 min to allow DNA–NTR II complexes to form.
  5. Add the DNA–NTR II complexes dropwise onto HEK293T cells cultured in 1.8 mL DF-12 + 2% FBS in the dish.
  6. Return cells to the incubator (37 °C, 5% CO₂) and culture under standard conditions.

*Notes:

Serum condition: Although the manufacturer recommends forming complexes in serum-free medium, in our hands NTR II–DNA complexes prepared and used directly in DF-12 + 2% FBS yielded successful transfection in HEK293T. If efficiency drops or cytotoxicity increases, switch to serum-free medium for complex formation and/or reduce serum during the first 4–6 h, then return to DF-12 + 2% FBS.

Protein Experiments

In this protocol, you can find how to lyse cells prior to SDS–PAGE/Western blot.

Materials:

  • NETN lysis buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris‑HCl pH 8.0, 0.5% NP‑40)
  • Protease & phosphatase inhibitor cocktails
  • Ice, microtubes, centrifuge

Protocols:

  1. Wash cells with cold PBS; add NETN and inhibitors on ice.
  2. Vortex and incubate 5 min on ice, repeat 3 times.
  3. Spin 12,000 rpm, 15 min, 4 °C; collect supernatant as lysate for quantification.

In this protocol, you can find how to lyse cells prior to SDS–PAGE/Western blot using freeze–thaw cycles.

Materials:

  • PBS (ice-cold)
  • Protease & phosphatase inhibitor cocktails
  • Dry ice/ethanol bath or −80 °C freezer
  • 25 °C dry bath
  • Microtubes, centrifuge

Protocols:

  1. Wash cells with cold PBS and collect into a microtube. Add protease/phosphatase inhibitors.
  2. Immerse the tube in liquid nitrogen for 10–20 sec to freeze cells completely.
  3. Thaw the tube in a 25 °C dry bath for 30–60 sec until fully liquid.
  4. Repeat steps 2–3 for a total of 10 cycles.
  5. Centrifuge at 12,000 rpm, 15 min, 4 °C to pellet debris.
  6. Collect the supernatant as lysate for quantification or downstream analysis.

In this protocol, you can find how to concentrate culture supernatant (sup) using centrifugal filter units for downstream protein analysis.

Materials:

  • Collected culture supernatant (after PBS wash and Opti-MEM incubation)
  • Centrifuge tubes (15 mL, sterile)
  • 0.45 µm sterile syringe filter
  • Amicon Ultra-0.5 centrifugal filter units, 3 kDa MWCO
  • Tabletop centrifuge (fixed-angle rotor, capable of ≥14,000 × g)
  • Ice

Protocols:

  1. Collect culture supernatant and centrifuge at 1500 × g for 5 min to remove cell debris.
  2. Filter the clarified supernatant through a 0.45 µm filter to remove remaining cells and large particulates.
  3. Transfer clarified supernatant into an Amicon Ultra-0.5 (3 kDa) centrifugal filter unit.
  4. Centrifuge at 14,000 × g, 4 °C until the desired final volume is reached (e.g., concentrate from 500 µL to 100 µL), usually 15-20 mins.
  5. Recover concentrated supernatant from the filter unit by inverting the column into a clean tube and centrifuging at 1,000 × g for 2 min.
  6. Keep concentrated samples on ice for immediate use or store at –80 °C.

In this protocol, you can find how to quantify protein concentration using a BCA assay at 562 nm.

Materials:

  • BCA Protein Assay Kit (working reagent)
  • BSA standards (serial dilution)
  • 96‑well clear plate; plate reader (λ = 562 nm)

Protocols:

  1. Prepare BSA standards and dilute lysates in the same buffer.
  2. Add X µL sample/standard + Y µL BCA working reagent per well (follow kit).
  3. Incubate 37 °C, 30 min; read A562.
  4. Generate standard curve; calculate sample concentrations.

In this protocol, you can find how to prepare quantified lysates for electrophoresis.

Materials:

  • 5 × Laemmli sample buffer (Tris-HCl pH6.8, SDS, β-ME, glycerol, Bromophenol Blue)
  • NETN or PBS

Protocols:

  1. Mix protein lysate with 5× sample buffer to a final 1× concentration, and use NETN/PBS to adjust volumes so that all samples have the same final volume.
  2. Heat at 95 °C for 5 min to denature proteins.
  3. Cool on ice and briefly spin down.
  4. Load ~20 µg protein per lane for SDS–PAGE
  5. If the gel will be run the next day, store prepared samples at −20 °C until use.

In this protocol, you can find how to separate proteins by size using SDS–PAGE.

Materials:

  • Protein samples
  • hand-cast SDS–PAGE gels (12% resolving gel with 5% stacking gel)
  • Protein ladder (molecular weight marker)
  • Running buffer (1× Tris-Glycine-SDS)
  • Electrophoresis apparatus and power supply

Protocols:

  1. Assemble SDS–PAGE gel in electrophoresis tank and add 1× running buffer.
  2. Load protein samples and markers into wells.
  3. Run at 85 V through stacking gel, then increase to 130 V until the dye front reaches the bottom.
  4. Stop electrophoresis and prepare for transfer.

In this protocol, you can find how to transfer proteins to a membrane and detect them by antibody-based chemiluminescence.

Materials:

  • PVDF (requires methanol activation)
  • Transfer buffer
  • Filter papers and sponges
  • Blocking buffer (5% milk in TBST)
  • Primary antibody (e.g., p-STAT3 Y705, His-tag…)
  • Loading control antibody (e.g.,GAPDH)
  • HRP-conjugated secondary antibody
  • TBST (TBS + 0.1% Tween-20)
  • ECL substrate
  • Imaging system (chemiluminescence detection)

Protocols:

  1. Assemble transfer sandwich (sponge–filter paper–gel–membrane–filter paper–sponge).
  2. Transfer proteins to the membrane (e.g., wet transfer at 100 V for 150 min , 4 °C).
  3. Block membrane with 5% milk in TBST for 1h at room temperature.
  4. Incubate with primary antibody (overnight at 4 °C).
  5. Wash membrane 3 × 6 min with TBST.
  6. Incubate with HRP-conjugated secondary antibody (60 min at RT).
  7. Wash membrane 3 × 6 min with TBST.
  8. Add ECL substrate and detect bands with imaging system.

In this protocol, you can find how to detect His-tagged anti-IL-23 scFv by ELISA.

Materials:

  • High-binding 96-well ELISA plate
  • ELISA coating buffer
  • Recombinant IL-23 (5 ng/well in 50 µL coating buffer)
  • PBS-T (PBS + 0.1% Tween-20)
  • Blocking buffer: 5% milk in PBS
  • His-tagged scFv samples (serial dilutions in PBS-T)
  • Mouse anti-His antibody (1°; ~1:500 in 5% milk)
  • Anti-mouse-HRP antibody (2°; 1:5000 in 5% milk)
  • TMB substrate (100 µL/well)
  • 1 N HCl (50 µL/well)
  • Plate reader (OD 450 nm)

Protocols:

  1. Coat wells with IL-23 (5 ng/well in 50 µL coating buffer) and incubate overnight at 4 °C.
  2. Remove coating solution and wash wells 3× with 200µL PBS-T per well (1 min each).
  3. Add 100 µL/well blocking buffer (5% milk in PBS), incubate 37 °C for 1 h.
  4. Remove blocking solution, and wash wells 3× with 200µL PBS-T per well .
  5. Add 50 µL/well scFv samples (serial 2-fold dilutions, e.g. 5 → 2.5 → 1.25 → 0.625 → 0.3125 µg/mL), incubate 37 °C for 1 h.
  6. Wash wells 3× with 200µL PBS-T per well.
  7. Add 50 µL/well mouse anti-His antibody (diluted ~1:500 in 5% milk), incubate 37 °C for 1 h.
  8. Wash wells 3× with 200µL PBS-T per well.
  9. Add 100 µL/well anti-mouse-HRP antibody (1:5000 in 5% milk), incubate 37 °C for 1 h.
  10. Wash wells 3× with 200µL PBS-T per well.
  11. Add 100 µL/well TMB substrate, incubate RT for 5–15 min until color develops.
  12. Add 50 µL/well 1 N HCl to stop the reaction.
  13. Measure absorbance at 450 nm.