Protein
SDS-PAGE gel recipe
Work out the component volumes for casting SDS-PAGE gels. Set the resolving and stacking percentages and volumes and how many gels you are pouring; the tool scales a standard Laemmli recipe, with water as the balance.
Resolving gel
| Component | Volume |
|---|
Stacking gel
| Component | Volume |
|---|
A standard Laemmli recipe. Water is the balance after the other components, so add it first, then the acrylamide, buffer and SDS, and add APS and TEMED last since they start polymerisation. Degas or work quickly. Acrylamide is a neurotoxin before it sets — handle the unpolymerised solution with gloves.
Formula
acrylamide = volume × (%T ÷ stock %); water = volume − all other components
- %T
- the total acrylamide percentage of the gel
- stock %
- the concentration of your acrylamide/bis stock, usually 30%
- resolving buffer
- 1.5 M Tris-HCl pH 8.8 at one quarter of the volume
- stacking buffer
- 1.0 M Tris-HCl pH 6.8 at one eighth of the volume
Worked example
A 10% resolving gel, 10 ml
- %T 10
- stock 30%
- volume 10 ml
- Acrylamide: 10 × (10 ÷ 30) is 3.33 ml.
- Resolving buffer: a quarter of 10 is 2.5 ml.
- Water: the balance, about 4.0 ml.
Answer 3.33 ml acrylamide, 2.5 ml buffer, ~4.0 ml water, plus SDS, APS and TEMED
Common mistakes
- Adding APS and TEMED too early. They start polymerisation, so add them last and pour promptly, or the gel sets in the tube.
- Using the wrong buffer for each layer. The resolving gel needs pH 8.8, the stacking gel pH 6.8; swapping them ruins the stack and the band sharpness.
- Handling unpolymerised acrylamide without gloves. The monomer is a neurotoxin; only the set gel is safe to handle bare-handed.
Frequently asked
Which percentage should I use?
Lower percentages resolve large proteins, higher percentages small ones. A 10% gel is a good all-rounder; go to 12–15% for small proteins and 7.5% for large.
Why is my gel not polymerising?
Old APS is the usual culprit — make it fresh or keep frozen aliquots. Too little TEMED or a cold room also slows it.
Can I make a gradient gel with this?
No, this pours a single percentage per layer. A gradient needs two mixes and a gradient maker, which is a different workflow.