SDS-PAGE Gel Percentage & Recipe Calculator
Pick the acrylamide percentage that resolves your protein (or two proteins), then get resolving and stacking gel recipes from a 30% or 40% stock: Tris, SDS, APS, TEMED and water. Sambrook or Bio-Rad conventions, scaled to your gel count and cassette size.
How it works
mix volume = volume per gel × gels × (1 + extra) · acrylamide stock = volume × gel % ÷ stock % · resolving: 25% v/v 1.5 M Tris pH 8.8, 1% v/v 10% SDS, 10% APS 1% (Sambrook) or 0.5% (Bio-Rad), TEMED 0.04–0.08% or 0.05% · stacking: 1.0 M Tris pH 6.8 at 12.5% v/v (Sambrook, 5% gel) or 0.5 M at 25% v/v (Bio-Rad, 4% gel) · water to volume
Polyacrylamide pore size is set by the acrylamide concentration. A higher percentage sieves small proteins apart but traps large ones near the top, so choose the resolving gel that runs your protein to roughly the middle. The calculator picks from a guide of separation ranges. Under More options you can add a second protein, and it finds a single percentage that covers both or tells you to use a gradient gel. The discontinuous Laemmli system first stacks the sample into a thin band in a low-percentage, pH 6.8 gel, where glycine ions move slowly, then separates it in the pH 8.8 resolving gel. Each component is a fixed share of the final volume, so the recipe scales to any number of gels and any cassette. Choose a mini-gel size or type the volumes, and pick the Sambrook convention (1% APS, 5% stack in 1.0 M Tris) or the Bio-Rad one (0.5% APS, 4% stack in 0.5 M Tris). The acrylamide volume follows from your stock, 30% or 40%. Ammonium persulfate supplies free radicals and TEMED speeds their formation, so both go in last. A 10% extra covers pipetting losses and the meniscus.
Worked example
For a 55 kDa protein the auto pick is a 10% gel. Two mini gels at 5 mL each, plus 10%, make 11 mL. That takes 11 × 10 ÷ 30 = 3.67 mL of 30% acrylamide, 2.75 mL of 1.5 M Tris pH 8.8, 110 µL of 10% SDS, 110 µL of 10% APS, 4.4 µL of TEMED and 4.36 mL of water. The 4.4 mL of 5% stacking gel takes 0.73 mL of acrylamide, 0.55 mL of 1.0 M Tris pH 6.8, 44 µL each of SDS and APS, 4.4 µL of TEMED and 3.02 mL of water. With a 40% stock, the resolving gel needs only 2.75 mL of acrylamide and the water rises to make up the difference.
Sources: Bio-Rad — A Guide to Polyacrylamide Gel Electrophoresis and Detection (Bulletin 6040) · Bitesize Bio — SDS-PAGE gel recipe
Frequently asked questions
What percentage for a 15 kDa protein?
15%, or a 16.5% Tris-tricine gel for anything below about 10 kDa, where standard glycine gels lose resolution.
My gel will not set — why?
Old APS (make it fresh weekly), too little TEMED, oxygen (degas or overlay the resolving gel), cold reagents or a low-percentage gel that simply needs 45–60 minutes.
Can I use a 40% acrylamide stock?
Yes. Only the acrylamide volume changes: it is the final volume × gel % ÷ 40 instead of ÷ 30, and the water makes up the rest. Set the stock under More options. The bis ratio (37.5:1, 29:1 or 19:1) does not change the volumes, but a higher bis share gives smaller pores.
What is the difference between the Sambrook and Bio-Rad recipes?
Both use 0.375 M Tris pH 8.8 and 0.1% SDS in the resolving gel. Sambrook uses twice as much APS (0.1% final) and a 5% stacking gel made with 1.0 M Tris pH 6.8. Bio-Rad’s handcasting recipe uses 0.05% APS and TEMED and a 4% stack made with 0.5 M Tris pH 6.8. Both give 0.125 M Tris in the stack, and both work.
How do I resolve two proteins of very different size?
Enter the second protein under More options. If both fit one percentage’s range, that percentage is chosen. If not, for example 15 and 150 kDa, a 4–20% gradient gel separates both on one run.
Related calculators
Standard laboratory formulas and textbook recipes (Sambrook, Bio-Rad, Thermo Fisher protocol conventions). Follow your own kit or instrument manual where it differs, and validate with controls — this tool does not replace lab training or safety procedures.