I am preparing the SDS PAGE gel and unfortunately it did not polymerise. I already used the freshly prepared APS and TEMED. I'm also prepared the fresh buffers and checked their pH.
Is anyone can suggestion why this still happen and how to solve it.
Thanks.
Hi, only a question, the solution volume that remain when you prepare a handmade gel, did it start to polymerize? If you are sure of your reagents freshly prepared,maybe the problem colud be the temperature. Some of my coworkers put the apparatus for gel polymerization in ah hoven at 37°C; you can try, first, to pepare the solution for the gel in a tube and put that in an hoven (or incubator at 37°C) and see if polymeraze.
If it not happen check the APS, if it is too old, or check the protocol if you add the correct amount of all the components
APS and temed are the usual culprits so get some temed from another lab/stock in case the stock bottle is too old. You must keep all oxygen away from the setting gel so cover the exposed areas. Check also that you are using an acrylamide/bisacrylamide mixture and not just acrylamide. make up a little mixture and put in plastic 1ml capped tube. In one tube add right aps and 2x too much temed and in the other 2x too much aps and right amount of temed cap it and leave to set to see if either of these reagents are defective. Check also the age of the acrylamide solution if you are using pre dissolved acrylamide in case of some polymerisation or white residue of precipitating acrylamide/salts/acrylic acid
What is the concentration of APS that you are using? What is the amount of TEMED that you are adding and also the solution volume of running and stacking buffer that you are preparing?
You may use APS and TEMED with more two, three folds. I faced your trouble three months ago. I think your APS had oxidized and could not make your gel polymerized.
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