SDS-PAGE resolving gel (8, 10, 12, 15%) recipe
Composition of the acrylamide resolving gel. Pick the percentage that suits your protein size and the amounts update. 10 mL casts about two 1.0 mm mini gels.
| Component | Amount | Final concentration (1X) |
|---|---|---|
| Water | 4.6 mL | |
| 30% acrylamide/bis-acrylamide (29:1) | 2.7 mL | 8% |
| 1.5 M Tris-HCl pH 8.8 | 2.5 mL | 375 mM |
| 10% SDS | 100 µL | 0.1% |
| 10% APS (fresh) | 100 µL | 0.1% |
| TEMED | 4 µL | 0.04% |
| Water | 4 mL | |
| 30% acrylamide/bis-acrylamide (29:1) | 3.3 mL | 10% |
| 1.5 M Tris-HCl pH 8.8 | 2.5 mL | 375 mM |
| 10% SDS | 100 µL | 0.1% |
| 10% APS (fresh) | 100 µL | 0.1% |
| TEMED | 4 µL | 0.04% |
| Water | 3.3 mL | |
| 30% acrylamide/bis-acrylamide (29:1) | 4 mL | 12% |
| 1.5 M Tris-HCl pH 8.8 | 2.5 mL | 375 mM |
| 10% SDS | 100 µL | 0.1% |
| 10% APS (fresh) | 100 µL | 0.1% |
| TEMED | 4 µL | 0.04% |
| Water | 2.3 mL | |
| 30% acrylamide/bis-acrylamide (29:1) | 5 mL | 15% |
| 1.5 M Tris-HCl pH 8.8 | 2.5 mL | 375 mM |
| 10% SDS | 100 µL | 0.1% |
| 10% APS (fresh) | 100 µL | 0.1% |
| TEMED | 4 µL | 0.04% |
Steps
- Mix water, acrylamide, Tris and SDS.
- Add APS and TEMED last, swirl gently and pour immediately between the plates (leave ≈1.5 cm for the stacking gel).
- Overlay with isopropanol or water to level the surface and let it set for 20–30 min.
- Pour off the overlay, rinse with water and add the stacking gel.
Good to know
- Choosing %: 8% for large proteins above 100 kDa, 10–12% for most proteins, 15% for those under 20 kDa (histones, LC3). Separation is best when your protein runs near the middle of the gel.
- Setting time depends on temperature and APS freshness: too fast → less TEMED; not setting → make fresh APS.
- To resolve two proteins of similar size (e.g. phospho- and non-phospho forms), use a lower percentage and run longer.
⚠ Safety Unpolymerised acrylamide is a neurotoxin — wear gloves and prefer ready-made solutions to powder. Polymerised gel is much safer.
FAQ
When is a gradient gel (4–20%) better?
When proteins of very different sizes must be seen together or the size is unknown. They are hard to cast, so most labs buy them.
How much for 1.5 mm or larger gels?
A 1.5 mm mini gel takes about 7.5 mL of resolving gel. Change the final volume in the calculator and every component scales.
References
- Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–685.
- Green MR, Sambrook J. Molecular Cloning: A Laboratory Manual. 4th ed. Cold Spring Harbor Laboratory Press; 2012.
- Harlow E, Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press; 1988.
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.