How to Make an SDS-PAGE Gel

How the gel works

SDS coats proteins with negative charge in proportion to their length, so in an electric field they move through the polyacrylamide mesh according to size alone. The gel has two layers: a short stacking gel at low pH and low percentage that squeezes each sample into a thin band, and a resolving gel below it that separates the proteins.

Choose the percentage

• 8%: about 40 to 200 kDa
• 10%: about 20 to 150 kDa
• 12%: about 15 to 100 kDa, a common general choice
• 15%: about 10 to 70 kDa

The stacking gel is usually 4% or 5%.

What each ingredient does

• Acrylamide/bis: forms the gel mesh; its percentage sets the pore size.
• Tris-HCl: pH 8.8 at 0.375 M in the resolving gel, pH 6.8 at 0.125 M in the stacking gel. The pH difference is what makes the samples stack.
• SDS: 0.1%, keeps proteins denatured and charged.
• APS and TEMED: start polymerization. Add them last, because the gel begins to set within minutes.

Pour the gels

1. Assemble the glass plates and check them for leaks with water.
2. Mix the resolving gel, add APS and TEMED, swirl, and pour to about 1.5 cm below where the comb's teeth will reach.
3. Overlay with isopropanol or water for a flat edge, and let it set for 20 to 30 minutes.
4. Pour off the overlay, mix the stacking gel with its APS and TEMED, pour it on top and insert the comb.
5. Let it set, then remove the comb and rinse the wells.

Unpolymerized acrylamide is a neurotoxin: wear gloves and handle the solutions with care.

Calculators

The SDS-PAGE Gel Calculator lists every volume for the resolving and stacking gels at any percentage and number of gels. To load equal amounts of protein, measure your samples with the Protein Concentration Calculator.