I am trying to lab-cast an SDS-PAGE gel for the electrophoresis of human serum proteins. Despite several attempts, I have failed get my gel to set. I have achieved some degree of polymerization, but the solute does not become firm enough for the comb to leave an impression. I am using the old-style Perspex 'flat-bed' green electrophoresis apparatus, which is all we have in our lab. The TEMED I am using has been opened for over two years; would this affect my result? Do you think fresh TEMED will work? Please help! Thank you, GiNgA xx

I don't do SDS-PAGE but I do DGGE which requires me to set up polyacrylamide gels.. I use fairly fresh TEMED and due to the amount of gels we run in our lab it rarely gets more than two months old.. I take it you are usng APS (Ammonium persulphate) for the polymerisation aswell? I make up fresh APS solution weekly as it is more important that this is fresh.. Also I store them both in a fridge to keep them fresher.. Hope this helps

Thank you Tricky, much appreciated. Yes, I am using ammonium persulphate (1.0g), sodium dodecyl sulphate (1.0g), acrylamide (10.0g), dH2O (100 mL) and N,N,N',N'-TEMED (100 uL). I tried using more ammonium sulphate and more TEMED, but no difference in quality. I reckon the TEMED is too old, but I also read that oxygen can inhibit polymerization, which could also be a factor. Thanks again Gx.

I use Tris buffer for the upper and lower buffer-is this just a standard gel for running proteins, or something more specialised?
Also when I demonstrate for undergrads the lecturer does a speech about acrylamide is dangerous and the ready made solutions (30 or 40% e.g. BioRad) are safer. He then goes on to debate whether that is true, but says the undergrads definately shouldn't use the powder in their 3rd year projects. Our lab always uses the read-made solutions. It is the APS and temed that causes the gel to set, it is liquid until I add these, then gets hot to the touch. Do you mix in a tube before pouring-i.e. is it not mixed thoroughly maybe? (Is the acrylamide % high enough?)When I make a gel I have a 16% resolving gel and upper stacking gel. I mix the reagents, pour between the plates and put a solvent on top, which I remove before pouring the top gel which sets around the comb so it is not open to air at the top.

Try this site
http://www.protocol-online.org/
The only way to see if it will work is to try it-maybe borrow some temed from a friend in another lab that they know works.

have you thought about buying the ready-gels from Bio-rad? much easier!

Thanks everyone, I really appreciate your input. I have read about stacking gels cc, but for now I am just interested in actually setting a single discontinuous gel to separate-out proteins in the sera samples. You are right about the toxicity of the reagents; they are unquestionably hazardous. My solution will be to use fresh TEMED, as my other chemicals are new. If that doesn't work, I will buy-in my gels ready made. I just wondered if anyone else had experienced this problem. Thanks again! GiNgAx