4% SDS-PAGE stacking gel recipe
The 4% stacking gel poured on top of the resolving gel; it concentrates samples into thin bands. 5 mL covers about two mini gels.
🧮 Volume calculator
| Component | Amount | Final concentration (1X) |
|---|---|---|
| Water | 3.05 mL | |
| 0.5 M Tris-HCl pH 6.8 | 1.25 mL | 125 mM |
| 10% SDS | 50 µL | 0.1% |
| 30% acrylamide/bis (29:1) | 665 µL | 4% |
| 10% APS (fresh) | 25 µL | 0.05% |
| TEMED | 5 µL | 0.1% |
Steps
- Mix water, Tris, SDS and acrylamide.
- Add APS and TEMED and pour onto the set resolving gel.
- Insert the comb immediately and let it set for 20–30 min.
Good to know
- After removing the comb, rinse wells with running buffer to clear unpolymerised acrylamide for straighter bands.
⚠ Safety Unpolymerised acrylamide is a neurotoxin — wear gloves and prefer ready-made solutions to powder. Polymerised gel is much safer.
FAQ
The stacking gel will not set.
Most likely old APS (10% APS lasts 1–2 weeks at 4 °C, longer frozen). Oxygen also inhibits polymerisation — do not mix vigorously.
This is a widely used standard composition. Amounts depend on the hydrate form and purity of your reagents — check the formula weight (FW) on the bottle and follow your institution's safety rules and the manufacturers' instructions.