Can anyone provide any insight as to why my Tris/Gly SDS-Gels are polymerizing slowly? This especially applies to my stacking gel (image attached) both 4% and 6%. The stacking gel wells polymerize slowly and are thin and lopsided.
-All buffers are kept at room temp (except 40% Acrylamide/bis-Aryclamide 37.5:1 kept at 4C)
-10% APS is made fresh
Thanks
We use TEMED stored at room temp for years and it always works. Make sure your APS is fine. Second, mixing the solution properly before putting it in the glass cassette might help. you can also try adding more temed and check in the tube whether it is solidifying. Mix the bottle of Acrylamide before use by gentle swirls. All the best. Also, recheck the amount of acrylamide/bis you are using from the standard protocols. Wait a little more for solidification, the left over gel mix in the tube can give an indication for it.
Thanks, Mohit Arora I have all new supplies (acrylamide, APS and TEMED) coming in incase something was out of date. I have also tried degassing with nitrogen gas or warming my buffer to increase polymerization speed. Both have not worked so hopefully with fresh reagents I can troubleshoot new ideas.
- You should degas the gel mixture either with vacuum (make sure that a magnetic flea is spinning in the solution before you apply the vacuum) or in an ultrasonic water bath. Oxygen is a radical scavenger and prevents polymerisation.
- The APS solution should be made daily and stored on ice.
- When mixing the APS and TEMED into the de-aerated gel mixture, avoid shaking air in again.
- Acrylamide solutions should be stored in the fridge over a spatula of anion exchanger to capture any acrylic acid that may form. Do not buy the solid acrylamide, it is very toxic, skin-permeable and such a fine powder that it would be difficult not to distribute some throughout your lab during weighing, especially for a beginner.
- Wells are easier to see if you add some phenol red to the stacking gel mixture (doi:10.1016/0003-2697(88)90297-7).
- Make sure that the top of your comb actually touches the mixture and thus prevents air access. In some designs there is a gap between the top of the comb and the top of the well-walls, leading to poorly formed walls. In that case, have new combs made from Teflon sheets in a craft shop (doi:10.1016/S0003-2697(02)00639-5).
- I have published a very detailed protocol for SDS-PAGE in doi:10.1007/978-1-61779-821-4_48, may be that is helpful.
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