How to use
- Pick the resolving gel percentage. The four listed options carry the protein size range the source recommends them for; choose “My own” to type any other percentage.
- Enter how many gels you are pouring. Nothing is shown until this box has a number in it.
- Pick the spacer thickness. The volume that fills a Bio-Rad Mini-PROTEAN cassette goes into the box beside it; for a large-format gel, type your own cassette volume there.
- Check the resolving share and the extra. The 75% and 10% defaults are ours, so adjust them if your comb is deeper or shallower than usual.
- Enter a protein size in kDa and the table below marks the percentages that cover it.
- Make up the two recipes in order. APS and TEMED go in last, immediately before pouring.
SDS-PAGE Gel Formula and practical notes
A polyacrylamide gel sets as acrylamide weaves a mesh, and the size of the holes in that mesh is what sieves the proteins. More acrylamide means a tighter mesh and better resolution of small proteins. The composition itself is a handful of ratios, which is why it scales cleanly to any volume.
One layer
Once the total volume is fixed, four reagents follow from it as ratios. Water is the remainder.
- 30% acrylamide/bis (mL)
- total × percentage ÷ 30
- Gel buffer (mL)
- total × 0.25
- 10% SDS (mL)
- total × 0.01
- Water (mL)
- total − acrylamide − buffer − SDS
- 10% APS (µL)
- total in mL × 5
- TEMED (µL)
- resolving: total × 0.5 · stacking: total × 1
How much to make
The cassette volume is split between the two layers, multiplied by the gel count, and the extra is added on top.
- Resolving gel (mL)
- cassette × share × gels × (1 + extra)
- Stacking gel (mL)
- cassette × (1 − share) × gels × (1 + extra)
Practical notes
- ⚠️ Acrylamide and bis-acrylamide are neurotoxins in solution. The risk drops sharply once the gel has set, but unpolymerised monomer can remain, so handle cast gels with gloves as well.
- The gel buffer differs between the layers: 1.5 M Tris-HCl pH 8.8 for the resolving gel, 0.5 M Tris-HCl pH 6.8 for the stacking gel. Swapping them stops the bands stacking.
- Make the APS fresh daily, and replace TEMED every three months — it oxidises and gradually loses catalytic activity, which is the source's own recommendation.
- Wrong catalyst concentrations show up as poor well formation or webbing behind the comb. The source's remedy is to raise the stacking gel's catalysts to 0.06% APS and 0.12% TEMED.
- The cassette volume table is Bio-Rad Mini-PROTEAN, and it is the volume that fills a cassette completely. The source notes that the amount actually needed varies with the comb and with whether a stacking gel is being cast.
- Large-format cassette volumes are not in the table: no primary document for them opened. Rather than publish a guessed figure, the box is left editable.
- The 75% resolving share and the 10% extra are our defaults, not the source's. Do not quote them as sourced figures.
FAQ
It follows from the size of the protein you want to see. Enter a size in kDa above and the table marks the percentages that cover it. Larger proteins want a lower percentage, smaller ones a higher percentage. For a sample spanning a wide range, a gradient gel beats any single percentage.
Yes. The recipe comes out at any percentage, because it is the same set of ratios throughout. What you do not get is a kDa range, because the source only lists four percentages and inventing the values in between is not something this page will do.
The stacking gel is not there to separate anything — it is there to concentrate the sample into a thin band at the face of the resolving gel. Large pores and a lower pH are what achieve that, so the percentage stays low and the buffer changes to 0.5 M Tris-HCl pH 6.8 instead.
The default is 10%. It covers what stays in the pipette and the beaker and what gets spilled while pouring, so the proportion can come down as the number of gels goes up. This figure is ours rather than the source's.
The causes the source lists are too little or too much APS or TEMED, failure to degas, too low a temperature, poor quality acrylamide, and old APS. Use APS made the same day, cast at room temperature, and degas for 10–15 minutes.
Often used together
Open →
Agarose Gel Calculator
Turns a gel volume and target percentage into agarose, buffer and stain amounts.
Open →
Western Blot Quantification
Normalises band densities and works out the fold change against the control.
Open →
Reagent Preparation Calculator
Calculates the stock and diluent volume needed for a target concentration and volume.