What are the factors that affect polymerization of resolving gel in SDS-PAGE electrophoresis for protein?
I have used 5 ml resolving gel 12% , (1.6ml) ddH2O/ (2ml) 30% acrylamide:bis- acrylamide/ (1.3 ml )1.5M Tris (pH 8.8)/ (50 ul) 10%SDS /(50ul) freshly prepared 10% APS, (4ul)TEMED. Which worked properly a week a go. But now it doesn't.
Is the problem that the gel solution did not polymerize? Degas the solution, and use fresh APS solution.
APS is hygroscopic. Make sure APS reagent powder which you are using has not absorbed moisture. Try using another fresh bottle of APS reagent powder and make freshly prepared 10% APS to prepare the resolving gel. Also TEMED should be stored in dark bottle at 4°C as it is light sensitive.
Try using fresh APS and TEMED, it should work.
Hi,.
As earlier suggested by others,either too little APS or TEMED or your APS is too old.Always use fresh APS. Also try and check your acrylamide quality.
Regards
As you mentioned that you prepared all the reagents just one week ago. So APS should work properly because it is not so old (if stored at 4C. Otherwise you have to try with fresh APS). TEMED could be the main reason after APS, make it sure it is not contaminated and/or expired, and should be kept in dark at 4C.
Thanks Adam, for your quick reply , actually Adam yes gel dosnt polymerize, and I always prepare APS at same day of my work.
Thanks Bhairavi , for your quick reply too..
Yes my APS absorbed some moisture, but i cganged it with new one completetly dry, actually TEMED is kept in 4C but not in dark bottle, may be the TEMED is the problem !!
Thanks Adesola for your quick reply.
I doubled the amount of TEMED, with no results at all, and my APS is fresh and new, i also changed the acrylamide and it was prepared yesterday.
Thanks tehmina, for your quick reply
As you.mentioned all sol. Are fresh , I think my TEMED is the problem, it was not kept in dark bottle, I will get new TEMED today and check the results , I will let you all know where the problem is...
Hi every one
My gel polymerized properly..after i did the following steps:
1. I used new dd H2O
2. I checked the pH of tris it was 8.6 , fixed to 8.8
3. Used another source for APS
4. The room was too cold , I put the mixture of gel in incubator 37 C for 20 min
5. There was no problem in TEMED it worked proberly , although it wasn't kept in dark bottle..now i put it in dark bottle..
Thanx
That sounds good Rana. So the main problem was temperature. Yes, below 25 gel doesn't polymerize. Good luck.
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