Aug 12, 2025
SDS PAGE
This protocol is a draft, published without a DOI.
- Shafik Oubihi1
- 1University College Dublin
Protocol Citation: Shafik Oubihi 2025. SDS PAGE. protocols.io https://protocols.io/view/sds-page-g7hubzj6x
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 12, 2025
Last Modified: August 12, 2025
Protocol Integer ID: 224532
Keywords: sds page gel, preparation
Abstract
Recipes for preparation of SDS PAGE gels.
Troubleshooting
Safety warnings
Unpolymerised acrylamide is a neurotoxin. Early signs of exposure include numbness, tingling, and tenderness to touch. Symptoms can be delayed several days to weeks and if exposure continues (even in small quantities), other symptoms may arise including excessive sweating, blue-reddish skin, peeling of skin, and weakness in limbs.
Wear gloves and work in a fume hood. Unpolymerised acrylamide can pass through unbroken skin. In the case of skin contact, remove any contaminated clothing and rinse the affected area with water and soap for at least 15 minutes.
Polymerised acrylamide is not toxic.
Before start
Prepare your gel casting rig. You can use water to test for leaks.
On your glass plate, mark a line 1 cm below where the bottom of your comb will be. This is the line that you will fill your separating gel to.
10% Separating Gel
This recipe is enough to cast two gels.
Add the following to a 15mL tube:
- 4.1 mL dH2O
- 2.5 mL 1.5 M Tris (pH 8.8)
- 3.3 mL Acrylamide (30%)
- 50 µL SDS
When you are ready to cast the separating gel, add 100 µL Ammonium persulphate (10% w/v) and 10 µL TEMED in quick succession. This will start the polymerisation.
Invert tube a few times to mix, but do not shake. Pipette the solution between the glass plates up to your fill line. Tap on the benchtop to remove any bubbles, and overlay with a layer of ethanol or isopropanol.
Once the gel has polymerised, discard the alcohol layer and rinse with running buffer or water. Use filter paper to remove excess from the top of the gel before pouring the stacking gel.
6% Stacking Gel
This recipe is enough stacking gel for two gels.
Add the following to a 15mL tube:
- 2.7 mL dH2O
- 0.5 mL 0.5 M Tris (pH 6.8)
- 0.8 mL Acrylamide (30%)
- 20 µL SDS
When you are ready to cast the stacking gel, add 40 µL Ammonium persulphate (10% w/v) and 4 µL TEMED in quick succession. This will start the polymerisation.
Pipette the solution between the glass plates and insert the comb. Check for any bubbles.