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SDS-PAGE-Calc Gel Recipe

Separating + stacking gels · by gel count and % · printable protocol

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Parameters

One 18×16 mm glass sandwich counts as one gel; at the default volumes a gel needs 8 ml of separating and 5 ml of stacking solution.

%

Typical: 8% (large proteins), 10% (general), 12%, 15% (small proteins). Higher per cent, smaller pores.

%

Labs usually run 30% (29:1 or 37.5:1) or 40% acrylamide/bis stock - type any concentration you have.

%

Stacking gels are almost always 4% (sometimes 5% or 6%); the pH 6.8 to pH 8.8 moving interface is what concentrates the sample.

ml

Match this to your casting stand and plates - 8~10 ml of separating solution is normal for a 0.75 mm mini gel.

ml

Recipe

Protocol

Choosing a percentage

Separating gel Fractionation range Typical use
8%above 100 kDaLarge proteins
10%40 - 200 kDaGeneral purpose, most antibody targets
12%15 - 100 kDaThe everyday default
15%10 - 70 kDaSmall to medium proteins
15% over 8%10 - 200 kDaTwo-layer gel, wide range in one run
20%3 - 50 kDaPeptides and very small proteins
Tris-Tricine gelbelow 10 kDaPeptides and cleavage products (needs the Tricine buffer system)
4% stacking-Used in almost every routine recipe

The table above is a rule of thumb, not something this calculator produces: the separating gel percentage sets the fractionation range, and pores shrink as the percentage rises.

What this tool is

SDS-PAGE-Calc does the mental arithmetic of gel pouring in one pass: enter a gel count and the separating and stacking percentages and it returns ddH2O, acrylamide stock, Tris, SDS, APS and TEMED volumes for both gels, followed by a six-step protocol you can copy or print. It mirrors cytographica.com/lab/acryl2.html formula for formula (8 ml of separating and 5 ml of stacking solution per gel, Tris at 1/4 of the volume, SDS and APS at 1/100, TEMED at 1/1000) and adds live recalculation, a custom stock concentration and volume per gel, and text export.

Everything is computed in your own browser: there is no backend, nothing is uploaded, and your parameters sit in local storage so a refresh restores them. The original site is a 2007 form that prints through a popup window; this is a static page with a print stylesheet, so it also works on a phone at the bench.

Features

How to use it

  1. Enter the number of gel sandwiches you are casting.
  2. Enter the separating gel percentage and pick or type the acrylamide stock; leave the stacking gel at 4%.
  3. Measure against the recipe table: ml for the bulk components, µl for SDS, APS and TEMED - the last two go in last.
  4. Hit "Copy recipe text" for your lab notebook, or "Print / save as PDF" to put the steps on the gel box lid.

FAQ

Why do the two gels use different pH?
The stacking gel is 0.125 M Tris pH 6.8, the separating gel 0.375 M Tris pH 8.8. At pH 6.8 glycine barely ionises, chloride runs ahead of it and the protein-SDS complexes are trapped in between, so the moving interface squeezes the sample into a band only microns thick; once that interface enters the pH 8.8 separating gel glycine fully charges and overtakes the proteins, and molecular sieving takes over. That concentration step is why it is called a stacking gel.
Why are APS and TEMED added last?
Together they generate the free radicals that polymerise acrylamide, so the clock starts the moment they go in and the solution thickens within minutes at room temperature. Mix and degas the water, stock, Tris and SDS first, set up the plates and running buffer, then add TEMED and APS and pour and comb immediately. A 10% APS solution only keeps for one to two weeks at 4 °C, and expired APS is the single most common reason a gel refuses to polymerise.
What changes between a 30% and a 40% stock?
Only the dilution starting point: for 10 ml of 10% gel you take 3.33 ml of 30% stock or 2.50 ml of 40% stock and top up with water - the final gel is identical. 30% (29:1 or 37.5:1) stays in solution better and is less viscous; 40% needs less volume and leaves more room for water. The bis cross-linking ratio (29:1, 37.5:1, 19:1) sets pore size and brittleness and is outside what this calculator covers.
Is the calculated volume enough for my plates?
The default 8 ml separating plus 5 ml stacking suits one 18×16 mm sandwich at 0.75~1.0 mm, so a four-gel Mini-PROTEAN Tetra run means entering 4. If the meniscus sits below the comb teeth, raise the separating volume per gel to 10 ml; scale the same way for 1.5 mm or larger formats. Better to have a couple of millilitres left over than to run out mid-pour.
Are my parameters uploaded?
No. The page is fully static with no backend; the arithmetic runs locally in your browser and the parameters only ever go to your browser's local storage. Nothing about your unpublished recipes leaves the machine.

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