Before you start
- Calculate the volume for equal protein per lane from your BCA results.
Reagents and materials
Totals are for 10 samples (+10% extra). Per-sample amounts are in the middle column.
| Reagent | Per sample | Total |
|---|---|---|
| Protein (10–40 µg lysate per lane) | 20 µg | 20 µg |
| 4× Laemmli sample buffer with reducing agentper 20 µL loading volume | 5 µL | 5 µL |
| Running buffer (1× Tris-glycine-SDS)per sample share of the tank | 50 mL | 50 mL |
| Prestained protein ladder | 0.5 µL | 0.5 µL |
Buffers and recipes
10× Tris-glycine-SDS running buffer per 1 L
| Tris base (250 mM) | 30.3 g |
| Glycine (1.92 M) | 144 g |
| SDS (1%) | 10 g |
| Ultrapure water | to 1 L |
Do not adjust pH (≈ 8.3). Dilute to 1×.
10% resolving gel per 10 mL (two mini gels)
| Water | 4.0 mL |
| 30% acrylamide/bis (37.5:1) | 3.3 mL |
| 1.5 M Tris-HCl, pH 8.8 | 2.5 mL |
| 10% SDS | 0.1 mL |
| 10% APS (fresh) | 0.1 mL |
| TEMED | 4 µL |
Gel %: 8% for > 100 kDa; 10% for 30–100 kDa; 12% for 15–60 kDa; 15% for < 20 kDa; or 4–20% gradient for broad ranges.
5% stacking gel per 3 mL
| Water | 2.1 mL |
| 30% acrylamide/bis | 0.5 mL |
| 1.0 M Tris-HCl, pH 6.8 | 0.38 mL |
| 10% SDS | 30 µL |
| 10% APS | 30 µL |
| TEMED | 3 µL |
4× Laemmli sample buffer per 10 mL
| 1 M Tris-HCl, pH 6.8 (250 mM) | 2.5 mL |
| SDS (8%) | 0.8 g |
| Glycerol (40%) | 4 mL |
| Bromophenol blue (0.02%) | 2 mg |
| β-Mercaptoethanol (10%; add before use) | 1 mL |
| Water | to 10 mL |
Or use 200 mM DTT instead of β-mercaptoethanol.
Equipment
- Gel casting set or precast gels
- Electrophoresis tank and power supply
- Heat block (95 °C)
Procedure
Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.
Cast the gel (if hand-casting)
Pour resolving gel, overlay with isopropanol or water, let set ~30 min; pour stacking gel and insert the comb.
Prepare samples
Mix protein with 4× sample buffer and water to equal volumes.
Denature
Heat at 95 °C for 5 min (or 70 °C for 10 min for membrane or aggregation-prone proteins), then spin down.
Load
Assemble the tank with 1× running buffer, rinse wells and load ladder and samples.
Run
Run at 80–100 V through the stacking gel, then 120–150 V until the dye front reaches the bottom.
Next step
Transfer for western blotting or stain with Coomassie.
Critical points
- Load equal protein and keep lane volumes similar.
- Avoid loading in edge wells or fill empty wells with sample buffer for even running.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Smiling bands | Lower voltage, keep the tank cool, use fresh buffer. |
| Smears | Load less protein, reduce salt or DNA (sonicate), denature fully. |
| Gel does not polymerise | Fresh APS, check TEMED, degas solutions. |
Safety
Unpolymerised acrylamide is a neurotoxin: gloves, no skin contact. β-Mercaptoethanol: fume hood.
Further reading
- Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (1970).
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.