1. Equipment

• Vertical electrophoresis system: power supply, gel tank, and associated accessories.
• Gel casting assembly: spacer plate (0.75/1.0/1.5 mm), short plate, and plastic cassette/frame.
• Sample loading & pipetting: micropipettes and tips (5 mL, 1 mL, 200 μL, 10 μL).
• Centrifugation & storage consumables: centrifuge tubes (1.5 mL, 15 mL, 50 mL); amber centrifuge tubes (for aliquoting TEMED).
• Glassware: beakers, volumetric flask (100 mL), graduated cylinders (100 mL, 500 mL, 1000 mL), glass rod.
• Reagent containers: wide-mouth bottles (500 mL, 1000 mL), amber bottles (250 mL, 500 mL).
• General consumables: filter paper, lint-free wipes, plastic wrap/sealable bags.
• Measurement & monitoring: pH meter, etc.

2. Reagents

Preparation (100 mL):

①Weigh acrylamide (Acr) 29.2 g and N,N′-methylenebisacrylamide (Bis) 0.8 g into a 100 mL beaker.

② Add 50 mL ultrapure water (warm water aids dissolution) and stir magnetically until fully dissolved.

③ Transfer to a 100 mL volumetric flask and bring to volume with distilled water to 100.0 mL.

④ Filter through medium-speed filter paper and store at 4 °C (light-protected recommended).

Notes:

① Storage & stability

• Acrylamide slowly deaminates to acrylic/diacrylic acid under light and alkaline conditions, affecting gel performance. After preparation, verify solution pH ≤ 7.0; store at 4 °C protected from light. Recommended shelf life ≤ 2 months.
• If insoluble matter appears (reagent impurities or precipitation during storage), filter before aliquoting for storage.

② Reagent purity & electrophoretic performance

• Acrylamide purity directly impacts resolution and background. Use electrophoresis-grade acrylamide and crosslinker (Bis).

③ Gel formation mechanism (overview)

• Acrylamide monomers polymerize via free-radical initiation into long chains; in the presence of N,N′-methylenebisacrylamide (Bis), a crosslinked network forms.
• Gel pore size depends on chain length and crosslink density: the monomer/crosslinker ratio, total monomer concentration, and reaction conditions (initiator, accelerator, temperature, pH) collectively determine separation range and resolution.

Preparation (100 mL):

① Weigh 18.17 g Tris into a 100 mL beaker.

② Add 80 mL distilled water and stir magnetically until fully dissolved.

③ Adjust to pH 8.8 by adding 6 mol/L HCl dropwise (monitor pH in real time).

④ Transfer to a 100 mL volumetric flask and bring to volume with distilled water to 100.0 mL; store at 4 °C (optional: filter, aliquot, and seal).

1.0mol/L Tris-HCl（pH6.8）

Preparation (100 mL):

① Weigh 12.11 g Tris into a 100 mL beaker.

② Add 80 mL distilled water and stir magnetically until fully dissolved.

③ Adjust to pH 6.8 by adding 6 mol/L HCl dropwise (monitor pH in real time).

④ Transfer to a 100 mL volumetric flask and bring to volume with distilled water to 100.0 mL; store at 4 °C (optional: filter, aliquot, and seal).

10% sodium dodecyl sulfate (SDS)

Preparation (100 mL):

① Weigh 10.00 g SDS into a 100 mL glass beaker (or 250 mL reagent bottle).

② Add ~80–90 mL ultrapure water and stir/vortex until completely dissolved.

③ Transfer to a 100 mL volumetric flask and bring to volume with ultrapure water to 100.0 mL; mix gently.

④ Store at room temperature; for long-term storage, keep at 4 °C protected from light. If crystallization occurs, redissolve with gentle warming (~37 °C).

Notes:

① Low-temperature crystallization & redissolution: SDS solutions crystallize easily at low temperature; warm (e.g., 37–50 °C) until crystals fully dissolve before use to avoid under-concentration.

② Preparation & storage suggestions: Avoid preparing very large batches for long storage; prepare in small batches or for short-term use to maintain concentration and performance.

10% ammonium persulfate (APS)

Preparation (10 mL):

① Weigh 1.00 g APS into a 15 mL centrifuge tube.

② Add ultrapure water to ~8–9 mL, dissolve with agitation, then bring to 10.0 mL.

③ Aliquot 1.0 mL per 1.5 mL tube, protect from light, and store at −20 °C.

Notes:

① Solid APS loses activity after opening due to hygroscopic deliquescence—prefer small packages to reduce repeated exposure.

② After opening a small package, prepare a single stock batch, then aliquot 0.5 mL per 1.5 mL tube; store long-term at −20 °C protected from light.

③ Thaw one aliquot per use; avoid repeated freeze–thaw. Do not return leftovers to the stock bottle to prevent contamination and activity loss.

TEMED (N,N,N′,N′-tetramethylethylenediamine)

Running buffer（10×Tris Glycine SDS Running Buffer，10×TGS）

Preparation (1000 mL):

① Weigh 30.28 g Tris and 144.13 g glycine into a 1000 mL beaker.

② Add 800 mL distilled water and stir magnetically until fully dissolved.

③ Transfer to a 1000 mL reagent bottle (or bring to volume in a cylinder then return); make up to 1000.0 mL with distilled water. Store sealed at room temperature; recommended use period ~1 month.

④ Dilute 10-fold with distilled water to prepare 1× working buffer as needed.

Note:

Verify pH of the stock after preparation; target is ~8.3. If deviation > 0.5 pH units, check water pH, reagent purity, and storage before making minor pH corrections.

Formulations for resolving and stacking gels

Separation Gel — 6%

Separation Gel — 8%

Separation Gel — 10%

Separation Gel — 12%

Separation Gel — 15%

Loading Gel（5%）

Sample loading buffer (Laemmli Loading Buffer)

Non-reducing sample buffer (5×):

Preparation (100 mL):

① Weigh 10.00 g SDS and 10 mg bromophenol blue into a beaker.

② Add 25 mL 1 mol/L Tris–HCl (pH 6.8) and stir to dissolve completely.

③ Add 50 mL glycerol and mix.

④ Transfer to a 100 mL volumetric flask and bring to 100.0 mL with ultrapure water.

⑤ Aliquot into 1.5 mL tubes and store at −20 °C (fully redissolve before use).

Reducing sample buffer (5×):

Non-reducing 5× buffer + 500 mM DTT

Preparation (1 mL):

Weigh 154 mg DTT and add ddH₂O to 1 mL. Store at −20 °C.

Notes:

① Choice and role of reducing agent: In the presence of SDS, use β-mercaptoethanol (2–5%, v/v) or DTT (final 50–100 mM) to cleave disulfide bonds and promote complete denaturation. Given β-mercaptoethanol’s strong odor and volatility, DTT is adopted here as the standard reducing agent.

② Timing of addition: Add freshly to the mixture of sample and loading buffer just before loading (mix and heat as needed) to ensure sufficient reducing activity.

③ Definition of final concentration: Stated concentrations refer to final composition in the 1× working mixture after combining sample with buffer.

④ Preparation & storage: 5× loading buffer can be stored at room temperature; dilute to 1× for use and add DTT fresh (do not premix long-term) to avoid reductant degradation.

Water: distilled water (dH₂O), ultrapure water (ddH₂O) or ultrapure (UP)

3. Polyacrylamide Gel Casting

(1) Gel cassette preparation

① Select plate thickness according to experimental needs.

② Wash plates with detergent and dry; then wipe with 75% (v/v) ethanol and rinse with distilled water; dry.

③ Before use, wipe again with lint-free tissue.

④ Assemble spacer plate and short plate per instructions and fix in the casting stand; check sealing and even clamping.

(2) Casting the resolving gel

① In a 50 mL centrifuge tube add, in order: distilled water, 30% Acr-Bis, 1.5 mol/L Tris–HCl (pH 8.8), and 10% SDS; mix gently.

② Add freshly prepared 10% APS and TEMED; mix quickly.

③ Using a 5 mL pipette, slowly dispense along the inner plate wall: fill until ~1.5 cm below the top of the short plate (for a 6 × 8 cm, 1.5 mm gel, typical volume ~3.75 mL).

④ Gently overlay with ~1 cm distilled water (fill the plate) to level the surface and exclude air.

⑤ Let stand at room temperature for 20–40 min; when a sharp interface forms with the overlay, polymerization is complete. Decant overlay water and wick away residual moisture with filter paper.

(3) Casting the stacking gel

① In a 50 mL tube add, in order: distilled water, 30% Acr-Bis, 1.0 mol/L Tris–HCl (pH 6.8), and 10% SDS; mix gently.

② Add fresh 10% APS and TEMED; mix immediately.

③ Slowly dispense along the inner plate wall to near the top; insert the comb vertically to form wells; allow 10–20 min to polymerize.

④ Use within ~30 min, or seal the gel (on the plates) in a zipper bag and store at 4 °C (add a small amount of distilled water in the bag to maintain humidity).

⑤ For use, mount the gel (on plates) in the electrophoresis tank, add running buffer, gently remove the comb, and prepare to load.

(4) Operating tips

① For mini gels, a 15 mL/50 mL centrifuge tube works well as a mixing vessel; mix by gentle rotation or repeated pipetting. For larger volumes, use an appropriately sized beaker and stir with a glass rod.

② Avoid creating bubbles; if microbubbles appear, guide them up the wall with a pipette tip before casting.

4. Protein Electrophoresis

(1) Sample preparation

① Pre-heat a water bath or dry block to 70–100 °C.

② Transfer the required amount of protein sample to 1.5 mL microtubes; label and record in order.

③ Add loading buffer at the indicated ratio; if using Laemmli 5× loading buffer, prepare at sample:buffer = 1:4 (v/v) (choose reducing or non-reducing as needed).

④ Heat in water/dry block for 5 min.

⑤ Cool to room temperature; quick-spin 10–30 s to collect liquid.

⑥ Arrange by label and keep ready for loading.

(2) Electrophoresis

① Mount the gel with wells facing up in the tank; add running buffer to both outer and inner chambers: the outer buffer should cover the electrodes/platinum wires; the inner buffer should fully submerge the wells.

② Remove the comb vertically and slowly; before loading, aspirate/dispense a small amount of buffer in each well to remove residual gel and bubbles.

③ Load samples with a micropipette in the preset order and record.

④ Run at constant 120 V; stop when the bromophenol blue front reaches the bottom of the gel, or adjust time as needed to achieve desired separation.

(3) Operating tips

① Use gel-loading tips; avoid touching the well bottom and prevent spillover into adjacent wells; control loading volume to avoid overloading and band distortion.

② Do not hold the tip too high above the well to avoid diffusion into buffer; complete loading consecutively to minimize dwell time of early-loaded samples in the wells.

References

1.Green MR; Sambrook J. Molecular Cloning: A Laboratory Manual (4th ed.). Chapter 19, Protocol 8: SDS–PAGE of proteins.

2.Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;277(7):680–685.

3.Davis B. Disc electrophoresis. II. Method and application to human serum proteins. Annals of the New York Academy of Sciences. 1964;121:404–427.

4.Ornstein L. Disc electrophoresis—I. Background and theory. Annals of the New York Academy of Sciences. 1964;121:321–349.

Aladdin: https://www.aladdinsci.com/