Protocols | iGEM Hamburg 2025

Introduction

Bacterial glycerol stocks are essential for the long-term preservation of plasmids and engineered strains. While plasmid DNA can be stored at -20°C, maintaining bacteria in glycerol allows you to easily recover the plasmid in its host strain without repeated transformations. The glycerol acts as a cryoprotectant, preventing damage to cell membranes during freezing, which keeps the bacteria viable for years at -80°C. This makes glycerol stocks a reliable way to build a strain library for iGEM projects.

Safety & Preparation

• Work in a clean area with sterile loops, pipette tips, and cryovials.
• Always wear lab coat and gloves.
• Use BSL-1 practices for non-pathogenic E. coli (e.g., DH5a, BL21).
• Clearly label all glycerol stock tubes with strain name, plasmid, antibiotic marker, and date.
• Avoid repeated freeze-thaw cycles, which decrease stock viability.

Materials

• Overnight culture of E. coli carrying plasmid of interest
• Sterile 50% (v/v) glycerol (autoclaved)
• Sterile ddH2O
• 2 mL screw-cap cryovials or tubes
• -80°C freezer

Protocol

Tips & Notes

• Always make at least two stocks per strain as a backup.
• Avoid storing at -20°C; bacteria may not survive long-term.
• If stocks repeatedly fail to recover, check glycerol concentration (final mix should be 25%).
• Keep an updated strain library list with stock location, plasmid map, and resistance markers.

Waste Disposal

• Dispose of used culture tubes, pipette tips, and loops in biohazard waste autoclave before discarding.
• LB cultures not used autoclave liquid waste before sink disposal.
• Wipe down work area with 70% ethanol after preparation.

Introduction

LB (Lysogeny Broth) medium is a standard nutrient-rich medium for growing E. coli and other non-pathogenic bacteria. LB agar plates are widely used for bacterial cloning, selection with antibiotics, and routine culture maintenance. Adding an appropriate antibiotic allows for selective growth of plasmid-containing bacteria. Proper preparation, sterilization, and storage of LB media ensure reproducibility and reliable experimental results.

Material & Reagents

• LB powder or pre-made LB medium
• Agar powder (for solid medium)
• Antibiotic stock solution (e.g., kanamycin 50 mg/mL)
• Distilled water
• Sterile Petri dishes
• Sterile containers for mixing medium
• Autoclave
• Sterile filtration setup (optional, for antibiotic addition)
• Bunsen burner or laminar flow hood (for sterile pouring)

Protocol

1. Prepare Medium

   For 15 Petri dishes, prepare 250 mL LB agar:

   • LB powder: 6.25 g (25 g/L)
   • Agar: 3.75 g (1.5% w/v)

   Dissolve powders completely in 250 mL distilled water in a suitable container.

2. Sterilization

   Autoclave at 121°C for 15-20 min.

3. Cool Agar

   After autoclaving, cool to 50-55°C (warm to touch but still liquid).

   Do not let the agar cool to room temperature before adding antibiotics, or it will solidify prematurely.

4. Add Antibiotic

   For kanamycin (final concentration 50 µg/mL in 250 mL):

   Volume of stock (50 mg/mL) = (50 µg/mL x 250 mL) / 50 mg/mL = 250 µL

   Add sterilely, e.g., via filtration or under a sterile hood.

   Mix gently to avoid bubbles.

5. Pour Plates
   • Pour ~15-20 mL per sterile Petri dish.
   • Work under sterile conditions (laminar flow hood or near a Bunsen burner).
   • Replace lids immediately after pouring.
6. Curing

   Allow plates to cure at room temperature for 20-30 minutes.

7. Storage
   • Store plates upside down (lid down) in sealed plastic bags or foil.
   • Keep in a refrigerator at 4°C.
   • Shelf life: ~2-4 weeks for kanamycin-containing plates.

Tips & Notes

• Ensure agar is completely dissolved before autoclaving to avoid uneven solidification.
• Add antibiotics only after agar has cooled to prevent degradation.
• Use sterile techniques when pouring plates to avoid contamination.
• LB plates without antibiotic can also be prepared for non-selective growth.
• Label plates with medium type, antibiotic, and preparation date.

Safety & Waste Disposal

• Follow BSL-1 practices for handling non-pathogenic E. coli.
• Dispose of leftover LB medium, tips, and contaminated materials in biohazard waste.
• Autoclave liquid or solid bacterial waste before disposal.
• Clean and decontaminate work surfaces with 70% ethanol after preparation.

Introduction

Overnight cultures of E. coli are a fundamental part of recombinant protein production in molecular biology. They provide a dense, actively growing bacterial population that can be used as a starting material for larger cultures, plasmid DNA extraction, or preparation of competent cells. Proper antibiotic selection and sterile techniques ensure healthy growth and prevent contamination.

Materials and Reagents

• E. coli colonies (strain of choice)
• LB medium (Luria-Bertani)
• Antibiotic stock solution (e.g., Kanamycin, 50 mg/mL)
• Sterile culture tubes (15 mL or 50 mL)
• Sterile pipette tips and micropipettes
• Agar plates with the appropriate antibiotic
• Shaking incubator (37°C, 220 rpm)

Preparation of Medium

Prepare antibiotic-containing LB medium:

• For 150 mL LB, add 150 µL of 50 mg/mL kanamycin stock (final concentration 0.05 mg/mL 1:1000 dilution).
• Mix thoroughly and keep sterile until use.

Inoculation & Culture

• Using a sterile pipette tip or loop, pick a single E. coli colony from an agar plate.
• Inoculate the colony into 4 mL of prepared LB-kanamycin medium in a sterile culture tube.
• Incubate overnight at 37°C with shaking at 220 rpm to ensure aeration and uniform growth.

The next day, the overnight culture is ready to use as a starter for larger cultures or plasmid preparation.

Tips & Notions

• Always pick a single colony to avoid mixed populations.
• Ensure the antibiotic is added after autoclaving and the medium has cooled to ~50-55°C to preserve activity.
• Avoid prolonged incubation (>16-18 hours), as overgrown cultures can reach stationary phase and reduce cell viability.
• Monitor culture turbidity; overnight cultures typically reach an OD600 of 2-3.

Safety & Waste Disposal

• Autoclave or treat any leftover culture with 10% bleach before disposal.
• Dispose of agar plates, pipette tips, and gloves that have been in contact with bacteria as biohazardous waste.
• Do not pour live cultures down the sink.

Introduction

IPTG (Isopropyl β-D-1-thiogalactopyranoside) is a molecular mimic of allolactose that binds the lac repressor and induces expression of genes under the control of the lac operon in E. coli. It is widely used to:

• Induce expression of cloned genes in lac-based expression systems (e.g., pET vectors).
• Screen blue/white colonies on X-gal plates.

Unlike natural inducers, IPTG is non-metabolizable, allowing for stable induction over time. Proper preparation of IPTG stock solutions and induction conditions ensures reproducible protein expression.

Materials & Reagents

• IPTG powder (≥99% purity, CAS 367-93-1)
• Distilled water (ddH2O)
• 0.22 µm syringe filter
• 15 mL Falcon tubes, sterile
• LB medium
• Kanamycin stock solution (50 mg/mL)
• Erlenmeyer flasks for bacterial culture
• E. coli BL21 or relevant expression strain
• Shaker incubator (25-37°C)
• Spectrophotometer (OD600 measurement)

Protocol

1. Prepare IPTG Stock Solution (1 M)
   • In a 15 mL sterile Falcon tube, add 3 mL ddH₂O.
   • Weigh and add 0.952 g IPTG, dissolve completely.
   • Adjust final volume to 4 mL.
   • Sterilize using a 0.22 µm syringe filter.
   • Aliquot 500 µL into sterile tubes and store at -20°C (stable up to 1 year).
2. Prepare E. coli Culture
   • In an Erlenmeyer flask, add LB medium.
   • Inoculate with overnight culture of E. coli BL21.
   • Add kanamycin at a 1:1000 dilution (final 50 µg/mL if stock is 50 mg/mL).
   • Incubate at 37°C 130 rpm until culture reaches OD600=0.4-0.6 (measure every 20-60 min).
3. IPTG Induction
   • Add the respective amount of 1 M IPTG to achieve a final concentration of 1 mM.
   • Incubate the culture at 25°C 120 rpm overnight for protein expression.

Tips & Notes

• Monitor OD600 closely; induction too early or too late can affect protein yield.
• Lower induction temperature (e.g., 25°C) often improves folding of recombinant proteins.
• Use sterile technique to avoid contamination during IPTG addition.
• Aliquot IPTG stock to minimize freeze-thaw cycles.

Safety & Disposal Waste

• Handle IPTG powder with gloves and avoid inhalation; it can irritate skin and eyes.
• Dispose of IPTG-contaminated tips, tubes, and media as biohazard waste if bacterial cultures are present.
• Decontaminate benches with 70% ethanol after use.
• Do not pour IPTG solutions down the drain; collect chemical waste according to institutional guidelines.

Introduction

Protein expression in the periplasm of gram-negativ bacteria (like E. coli) can be advantageous, as the oxidative environment facilitates the formation of disulfide bonds in proteins and helps with folding. Additionally, only few proteases are present in the periplasm. Proteins can be transported into the periplasm by adding specific signal sequences, for example the pelB-sequence to the N-terminus. To seperate the protein fraction present in the periplasm from the cell's cytosolic proteins, periplasmic extraction by osmotic shock can be performed. This process is based on the build-up of osmotic pressure on the outer membrane. Exposing the cells to buffer with lower solute concentration leads to rupture of the outer membrane, releasing the periplasmic fraction and leaving the cytosol of the cells intact. EDTA is used to weaken the integrity of the outer membrane, while sucrose prevents the cells from shrinking.

Preparation of TES buffer

for 50 mL of TSE buffer (100 mM Tris-HCI, 1mM EDTA, 500 mM sucrose):

• prepare 40 mL of dd H₂O in a measuring cylinder
• add 605.7 mg of Tris
• add 14.61 mg of EDTA
• add 85.6 g of sucrose
• stir with magnetic stir bar until dissolved
• adjust pH to 8 using HCI
• fill up to 50 mL with dd H₂O
• add 1/4 of a Protease Inhibitor Cocktail tablet (Sigma-Aldrich) and dissolve
• transfer into Schott glass bottle
• store at 4°C in fridge

Experimental procedure

• Spin 100 mL of liquid culture down at 3,234xg and 4°C for 30 min and discard supernatant (remove as much of remaining liquid as possible from centrifuge tube). Spin two times with 50 mL respectively when using 50 mL falcons.
• Carefully resuspend the cell pellet in 5 mL of TES buffer (100 mM Tris-HCI pH 8, 1 mM EDTA, 500 mM sucrose, 1/4 of protease inhibitor cocktail tablet). To avoid breaking the cells, use a sterile inoculation plastic loop to resuspend the pellet in the buffer before using a pipette tip.
• Incubate the suspension at room temperature for 10 min.
• Cold-shock cell by adding 5 mL of ice-cold sterile MQ water.
• Incubate suspension on ice for 10 min.
• Distribute suspension into 2 mL eppis if needed (the centrifuge fitting 50 mL falcons can only accelerate up to 3.234xg).
• Spin down the cells at 8.000xg and 4°C for 20 min and collect supernatant (contains the periplasmic extraction).
• Keep extraction on ice when working and at 4°C for storage.

Sample preparation for SDS-PAGE:

• mix 45 µL of supernatant containing periplasmic extraction and 5 µL of 10x SDS loading dye
• boil sample at 95°C for 5 min

Introduction

Sonication is a widely used method to lyse E. coli cells for protein extraction. Ultrasonic waves disrupt the bacterial cell membrane, releasing intracellular contents while keeping soluble proteins intact. Combined with centrifugation, this allows separation of soluble proteins from cell debris. Proper buffer composition, ice-cooling, and controlled sonication settings are essential to preserve protein activity and prevent overheating or denaturation.

Material & Reagents

• E. coli culture expressing protein of interest
• PBS (1x) for washing
• Sonication buffer:
  • 50 mM Tris-HCI (pH 8.0)
  • 500 mM NaCl
  • 20 mM Imidazole
  • 10% (v/v) Glycerol
  • ddH₂O to desired volume
• 15 mL sterile Falcon tubes
• Centrifuge capable of 3,234 x g at 4°C
• Ice bath
• Sonicator with adjustable power
• pH meter

Sonification Buffer Preperation

Mix all components in a 200mL Schott bottle.

• Set the pH value to 8.0 using NaOH or HCI.
• Keep buffer on ice before use.

Protocol

• 1. Cell Harvesting
  • Measure OD600 of the E. coli culture.
  • Normalize the volume of culture to be processed based on OD.
  • Centrifuge cells at 3,234 x g for 20 min at 4°C.
  • Discard supernatant.
• 2. Washing
  • Resuspend cell pellet in 1x PBS
  • Transfer to a 15 mL Falcon tube for washing.
  • Centrifuge again at 3,234 x g for 20 min at 4°C
  • Discard supernatant.
• 3. Cell Lysis via Sonication
  • Resuspend the cell pellet in 5-10 mL sonication buffer.
  • Keep samples on ice throughout the procedure.
  • Sonicate for 10 min total, 1x cycle, at 20% power.
• 4. Separation of Soluble Protein
  • Centrifuge lysate at 3,234 x g for 20 min at 4°C.
  • Carefully transfer the supernatant (contains soluble proteins) to a new 15 mL Falcon tube.
  • Discard cell debris.
  • Store protein-containing supernatant at -20°C.

Tips & Notes

• Keep samples on ice to prevent heat-induced protein denaturation.
• Sonication power and duration may need adjustment depending on cell density and sonicator type.
• Use freshly prepared buffer and avoid repeated freeze-thaw cycles of protein samples.
• Monitor protein concentration or activity after extraction to ensure integrity.

Safety & Waste Disposal

• Imidazole-containing buffer: dispose as liquid chemical/organic-aqueous waste; do not pour down the sink.
• Solid items (tips, gloves, filters) that contacted buffer: dispose as solid chemical waste.
• Any waste containing cells or protein should be treated as biohazardous if applicable, or autoclaved before disposal.

Introduction

Trichloroacetic acid (TCA) precipitation is a widely used method to concentrate proteins or remove contaminants such as salts, nucleic acids, and detergents from biological samples. TCA is a strong acid that lowers the pH, denatures proteins, and causes them to aggregate, making them insoluble. These aggregates can then be collected by centrifugation and further processed for downstream applications such as SDS-PAGE, Western blotting, or mass spectrometry.

When combined with sonication, TCA precipitation allows efficient recovery of proteins from complex samples while minimizing degradation.

Material & Reagents

• E. coli culture or protein-containing supernatant
• 50% TCA solution (see preparation below)
• Ice-cold acetone
• SDS-containing buffer.
• Tris-HCI (pH 6.8)
• SDS loading dye (10x)
• ddH₂O
• Centrifuge capable of 3,200-3,234 x g at 4°C
• 2 mL Eppendorf tubes
• 50 mL Falcon tubes
• Fume hood, gloves, goggles

Preparation of TCA solution

• Perform under a fume hood with gloves and goggles
• Add 30 mL ddH.O to a 50 mL Falcon tube.
• Weigh 28.93 g TCA and carefully dissolve in the water.
• Adjust volume to 50 mL with ddH.O.
• Store in a chemically resistant container.

Protocol

1. Harvest Cells and Clarify Supernatant
   • Centrifuge the culture at 3,234 x g for 20 min at 4°C.
   • Optionally, keep the cell pellet for other analyses.
   • Transfer supernatant to fresh Falcon tubes and centrifuge again at 3,234 x g, 4°C, 20 min to remove residual debris.
2. Protein Precipitation
   • Add the required amount of 50% TCA to achieve a final concentration of 10-15% in the sample.
   • Mix thoroughly.
   • Store at -20°C overnight for protein precipitation.
3. Pellet Proteins
   • Thaw samples at 4°C or on ice.
   • Centrifuge at 3,234 x g for 20 min at 4°C
   • Carefully remove supernatant into a designated acidic waste container.
4. Wash Protein Pellet
   • Add 400 µL ice-cold acetone and resuspend gently.
   • Centrifuge at maximum speed for 20 min at 4°C
   • Discard supernatant into halogenated organic waste.
   • Repeat washing once more.
   • Transfer the washed pellet into 2 mL Eppendorf tubes and centrifuge briefly.
   • Air-dry tubes for ~15 min to remove residual acetone.
5. Resuspend Proteins for Analysis

   Prepare SDS-containing buffer (1 mL total):

   • 200 µL ddH₂O
   • 700 µL Tris-HCI (pH 6.8)
   • 100 µL SDS loading dye (10x)

   Add 1 mL buffer to protein pellet and resuspend thoroughly.

   If solution appears yellow, sample is still acidic carefully add basic buffer (Tris-HCl, pH 8) until color turns blue (neutral).

Tips & Notes

• Keep all steps on ice or at 4°C to preserve protein integrity.
• Handle TCA and acidic supernatants under a fume hood.
• Avoid over-drying protein pellet; resuspend while still slightly moist for better solubilization.
• Precipitation efficiency may vary; optimize TCA percentage and incubation time if necessary.

Safety & Waste Disposal

• TCA solutions and supernatants: Collect in a chemically resistant container labeled "Halogenated Acidic Organic Waste (TCA)".
• Neutralization (if permitted): Can neutralize with NaOH to form sodium trichloroacetate, but it still counts as hazardous waste.
• Acetone washes: Dispose in the halogenated organic waste container.
• Protein pellets: After washing and drying, dispose as biological/solid lab waste according to institutional rules.
• Final step: All TCA-containing waste must be handled by your institution's hazardous waste disposal service.

Introduction

SDS-PAGE is a widely used method to separate proteins based on molecular weight. Proteins are denatured by SDS, a detergent that coats them with negative charges, ensuring that their electrophoretic mobility depends primarily on size, not shape or charge. Heating the sample further denatures proteins and allows uniform migration through the porous acrylamide gel. SDS-PAGE is essential in molecular biology for protein analysis, purity assessment, and subsequent applications such as Western blotting.

Material & Reagents

• Glass plates, casting frames, combs (10-or 15-well, 0.75-1.5 mm thickness)
• Acrylamide/Bis-acrylamide (30%)
• Tris-HCI (1 M, pH 6.8 and 1.5 M, pH 8.8)
• SDS (10%)
• APS (10% ammonium persulfate)
• TEMED
• Sample buffer (SDS + reducing agent + glycerol + bromophenol blue)
• Running buffer (Tris-Glycine-SDS)
• Coomassie Blue stain or alternative
• Protein ladder/marker
• ddH₂O, methanol
• Shaker/stirrer, fume hood, gloves, goggles

SDS-PAGE Preperation

An intact SDS PAGE electrophoresis system should include: a tank, lid with power cables, electrode assembly, cell buffer dam, casting stands, casting frames, combs (usually 10-well or 15-well), and glass plates (thickness 0.75mm or 1.0mm or 1.5mm). (We used mainly 15-well and 1.0 mm from Bio-rad brand)

The SDS PAGE gel in a single electrophoresis run can be divided into stacking gel and separating gel. Stacking gel (acrylamide 5%) is poured on top of the separating gel (after solidification) and a gel comb is inserted in the stacking gel. The acrylamide percentage in SDS PAGE gel depends on the size of the target protein in the sample.

Percentage and Volume of SDS-Gels

Volumes of stacking gel and separating gel differ according to the thickness of gel casting:

SDS-Gel Compositions

We used 12% and 15% acrylamid gels for the seperating gels.

Add the components for the 12% seperation gel in one flask, the 15% seperation gel in another flask and the 5% stacking also in another flask.

10X Sample buffer (loading buffer)

• SDS
• Dithiothreitol, or beta-mercapto-ethanol
• Glycerol
• Tris-HCl, pH 6.8
• Bromophenolblue

Make sure your target protein dissolved in the liquid phase, and no inappropriate ingredients present (e.g. guanidine hydrochloride can interact with SDS and cause precipitation) Generally, to treat your unprepared sample, you can use sonicator, lysis buffer or both to sufficiently make your target protein released, and centrifuge to make supernatant and pellet separated.

Running Buffer

10x SDS Running Buffer

Dilute the 10x SDS Running Buffer to 1x befor using.

(Approximately vol. of less than 1 liter is needed depending on the type of your electrophoresis system.)

Protocol

1. Make the separating gel:
   • Set the casting frames (clamp two glass plates in the casting frames) on the casting stands.
   • Prepare the gel solution (as described above) in a separate small beaker.
   • Swirl the solution gently but thoroughly.
   • Pipet appropriate amount of separating gel solution (listed above) into the gap between the glass plates.
   • To make the top of the separating gel be horizontal, fill in isopropanol into the gap until a overflow.
   • Wait for 20-30min to let it gelate.
2. Make the stacking gel:
   • Discard the water and you can see separating gel left.
   • Pipet in stacking gel untill a overflow.
   • Insert the well-forming comb without trapping air under the teeth. Wait for 20-30min to let it gelate..
3. Make sure a complete gelation of the stacking gel and take out the comb.
4. Take the glass plates out of the casting frame and set them in the cell buffer dam. Pour the running buffer (electrophoresis buffer) into the inner chamber and keep pouring after overflow untill the buffer surface reaches the required level in the outer chamber.
5. Prepare the samples:
   • Mix your samples with loading dye (40µL Sample and 10µL of the loading dye).
   • Heat them in thermocycler at 95°C for 5 min.
6. Load prepared samples:
   • Load samples into wells and make sure not to overflow.
   • Don't forget loading protein marker into the first lane.
   Then cover the top and connect the anodes.
7. Set an appropriate volt and run the electrophoresis.

   Voltage	Time
   80-100V	15-20 min
   120-140V	60-75 min.

8. As for the total running time stop SDS-PAGE running when the downmost sign of the protein marker (if no visible sign, inquire the manufacturer) almost reaches the foot line of the glass plate.

Tips & Notes

• Use fresh APS and TEMED for efficient polymerization.
• Avoid low temperatures during polymerization to prevent turbid gels.
• Ensure protein samples are fully soluble; remove interfering chemicals like guanidine.
• Higher acrylamide % separates small proteins better, lower % for large proteins.

Safety & Waste Disposal

Liquid Waste (buffers, gels in running buffer, wash solutions, etc.)

• Collect all SDS-containing solutions (sample buffer, running buffer, gel staining/destaining solutions if they contain SDS) in a dedicated, labeled container.
• Do not pour SDS solutions down the drain, because SDS is toxic to aquatic life and persistent in the environment.
• This waste is typically treated as halogen-free organic aqueous waste or detergent-containing chemical waste, depending on your institution.

Solid Waste (gels, pipette tips, gloves, paper towels contaminated with SDS)

• Place in a sealed plastic bag or container.
• Dispose of as solid chemical waste (not biological, unless your samples contain hazardous biological material too).

Staining/Destaining Waste

If you use Coomassie staining solutions with SDS present, collect them as SDS + dye-containing waste (special container, since the dyes are also hazardous).

Introduction

A Western Blot is a sensitive method to characterize proteins. Proteins are electrophoretically transferred from an SDS-PAGE onto a nitrocellulose membrane. Proteins can then be detected using labeled antibodies and chemiluminescence. Here, a monoclonal mouse antibody against 6x His-tags conjugated to the enzyme horseradish peroxidase was used to detect 6x His-tagged proteins.

Preparation of TBST buffer, blocking solution and transfer buffer

for 500 mL of 10x TBST buffer (200 mM Tris-HCI, 2 mM NaCl, 1% Tween 20):

• add 400 mL of dd H₂O to a 500 mL Schott bottle
• add 12.18 g of TRIS HCI
• add 40.03 g of NaCl
• adjust pH to 7.6
• add 5 mL of Tween 20
• fill up to 500 mL with dd H₂O

for 200 mL of blocking solution with 10% milk:

• add 20 mL of 10x TBST buffer to 180 mL of dd H₂O to a 500 mL Schott bottle
• add 19.97 g of milk powder and stir until dissolved

for 250 mL of 1x TBST buffer:

dilute 10x TBST buffer 1:10 (25 mL in 225 mL dd H₂O)

10x transfer buffer:

• 25 mM Tris base
• 190 mM glycine
• 20% methanol

Check pH and adjust to 8.3

for 1 L of 1x transfer buffer:

dilute 10x transfer buffer (100 mL in 900 mL dd H₂O)

Experimental procedure

• Run an SDS-PAGE to seperate proteins (do not stain gel after run).
• Soak blotting paper in 1x transfer buffer.
• Soak nitrocellulose membrane in methanol to regenerate it. Cut edge of nitrocellulose membrane to ensure correct orientation later.
• Place two sheets of blotting paper in cassette of Trans-Blot Turbo Transfer System, prevent drying out of paper by applying 1x transfer buffer.
• Remove SDS gel from chamber and cut off collection gel.
• Wash SDS gel 2x with dd H₂O
• Soak SDS gel and nitrocellulose membrane in 1x transfer buffer.
• Place nitrocellulose membrane onto blotting paper in cassette of Trans-Blot Turbo Transfer System.
• Place SDS gel onto nitrocellulose membrane.
• Soak two more sheets of blotting paper in 1x transfer buffer and place onto SDS gel in cassette of Trans-Blot Turbo Transfer System.
• Place the lid of the cassette on the stack.
• Run Western Blot at 1.3 A and 25 V for 20 min in Trans-Blot Turbo Transfer System.
• Remove membrane and place it in 1x TBST with 10% of milk for blocking.
• After 30 min of incubation, transfer membrane into fresh 25 mL of 1x TBST with 10% of milk. Add 12.5 µL of anti-His antibody conjugated to HRP (1:2000).
• Incubate overnight in cold room on shaker.
• Wash membrane shortly 2x with 1x TBST buffer.
• Wash membrane 2x for 5 min with 1x TBST buffer.
• Prepare chemiluminescent substrate by mixing 500 µL of Luminol/Enhancer and 500 µL of Stable Peroxide Buffer (from Thermo Fisher SuperSignal™ West Pico PLUS Chemilumineszenz-Substrat Kit)
• Place membrane onto plastic sheet (from clean cut-open trash bag).
• Add ca. 500 µL of luminol mix onto membrane. Fold plastic sheet over membrane to spread chemiluminscence mix, make sure that there are no bubbles.
• Measure chemiluminescence on ChemiDoc MP Imaging System (BIO-RAD).

Introduction

Liposomes are a widely used platform for drug and biomolecule delivery. Their biocompatibility, low immunogenicity, and ability to encapsulate both hydrophilic and hydrophobic molecules make them attractive for therapeutic applications. One of the most established approaches to fabricate liposomes is the Thin Film Hydration Method. This method is robust, reproducible, and allows control over lipid composition, size, and stability.

In this protocol, we outline the preparation of liposomes from phosphatidylcholine (PC) and cholesterol, using thin-film layer hydration, followed by freeze-thaw cycling to improve encapsulation efficiency and membrane uniformity.

Materials & Reagents

• Lipid stock solutions (e.g., PC, cholesterol) dissolved in organic solvent (chloroform or chloroform/methanol mix)
• Chloroform (analytical grade)
• HEPES buffer (10 mM HEPES, 150 mM NaCl, pH 7.5)
• Liquid nitrogen
• N₂ gas supply (for solvent evaporation)
• Glass tubes (round-bottom preferred)
• Rotary evaporator (optional, can be replaced by manual evaporation + N₂ stream)
• Exsiccator (for complete drying)
• Vortex mixer / orbital shaker
• Incubator/shaker (25-42°C)
• Extrusion device with polycarbonate membranes (100-200 nm)

Protocol

1. Thin Film Formation
   • Pipette 28 µL of PC and 9.1 µL of cholesterol into a glass tube.
   • Add 200 µL chloroform to dissolve the lipids completely.
   • Evaporate the solvent:
     • Gently heat the tube while swirling to form a thin, even lipid layer.
     • Apply a gentle N₂ gas stream to accelerate evaporation.
   • Place the glass tube in an exsiccator for ≥2 hours to remove residual solvent.
2. Rehydration
   • Add the required volume of HEPES buffer (10 mM HEPES, 150 mM NaCl, pH 7.5) to the dried lipid film.
   • Incubate at 42°C for 2 hours with gentle shaking (400 rpm).
   • Continue incubation overnight at 25°C, 400 rpm to complete hydration.
3. Liposome Formation

   Subject the lipid suspension to 10 freeze-thaw cycles:

   • Freeze rapidly in liquid nitrogen.
   • Thaw at 42°C with shaking (400 rpm).
   • Aliquot samples if needed.
   • Store at -80°C until use.
4. Size Homogenization

   Before experimental use, pass the liposomes through an extrusion membrane (100-200 nm pore size) or apply alternative size-reduction techniques (e.g., sonication, microfluidics) to achieve a uniform population.

Tips & Notes

• Ensure complete solvent removal during thin-film formation to prevent toxicity and instability.
• Keep lipids and solvents protected from light to avoid oxidation.
• Use sterile buffer if liposomes are intended for cell culture experiments.
• Control lipid ratios carefully: cholesterol stabilizes the bilayer but excessive amounts reduce flexibility.
• Extrusion through progressively smaller pore sizes improves reproducibility and polydispersity index (PDI).

Safety & Waste Disposal

• Chloroform and organic solvent waste must be collected separately in designated organic waste containers. Never dispose in the sink.
• Contaminated pipette tips, tubes, and consumables should be disposed of in chemical waste bins.
• N₂ gas should be handled in a well-ventilated area to avoid asphyxiation risk.
• Follow your institution's biosafety and chemical safety guidelines for storage and disposal of lipids and buffers.