I checked the pH of tris (6.8) and fresh preparation of APS solution. What is the reason?
What percentage APS and what percentage TEMED do you use?
Quick answer that I once got from a protein biologist was to double the APS when it does not polymerizes.
10% APS (30ul) (1g in 10ml DW) I add 5ul TEMED to make 3ml stacking gel
Those amounts seem fine, although you could try increasing the APS and see if it makes a difference. Another issue is exposure to oxygen. This inhibits polymerization, and some people degaze their solution before adding APS and TEMED. Set-ups like BioRad are designed to minimize oxygen exposure during polymerization by the shape of the combs. I generally let some stand in a glass pipet and see if it polymerizes in there. I know for sure that there is no oxygen coming in there. TEMED when it gets older and exposed to light might get bad. Beyond that, I am not sure.
sometime gel do not polymerizes at low tempearture, if you are preparing gel at low temperature, then, this might influence the polymerization process, As, I have also experienced such problem, and found that gel polymerizes best at 30-37 degree centigrade temperature.
if your TEMED or APS is old, use more of it or prepare fresh solutions. also check sds stock solution pH
Try to use an other source of DW.
Somebody had the same problem in my previous lab, he tried to prepare the gel with an other DW, and it just worked good.
What is the polyacrylamide % of your stacking gel? Also, is your polyacrylamide solution not too old?
Hope that helps!
Dear Sylvain Chauvet thank you. According to current protocol of molecular biology % of separation gel is 10% no certain concentration for stacking gel also the polyacrylamide solution is freshly prepared.
I came up with this problem once and found that APS is the main reason for that. When I changed the APS the gel properly polymerized.
Yes, Bahador is right, APS and also TEMED can be a problem. Is it possible that someone else in your department give you some for a gel. You will know if the problem come from here.
In addition to all of the suggestions above, I have also found that if after adding all the ingredients correctly, if there is a lot of mechanical force (ie vortexing to mix) that the polymerization is very poor. It may prevent crosslinking and/or introduce too much air into the mixture. So, I would always mix gently.
Check APS and TEMED. Old stock of the 2 items slowing down polymerisation
Al salamo alikom Dear Dr Serag these two PDF shows all related to SDS-PAGE and its artifacts or problems may appear during this test , these two PDF from BIO-RAD
In addition to problems with APS and TEMED, air also prevents polymerization, so make sure your gel casting apparatus is put together properly. You could try covering the gel with 70% ethanol after you insert the comb (use a pipette or squeeze bottle to put a thin layer of ethanol over the top of the comb, so it fills all the little cracks and crevices).
I usually cast my stacking gels at 4% polyacrylamide.
You must have fixed your problem a long time ago. You got really good suggestions above. I only wanted to add this because I'm just now encountering the same problem of poor polymerization: we forget sometimes that the fundamentals are in books. For example, I have a Proteins and Proteomics a Laboratory Manual by Richard J. Simpson and it offers a lot of basic information on how to prepare your reagents for SDS-PAGE, how to store them, how long they can/should be stored, etc. I feel like our troubleshooting should always start with those fundamentals.
Good luck!!
My stacking gel is not polimerizing and I'm using the same APS and TEMED for the running gel wich works well. I've heard in the lab that the tris-hcl solution must be prepared the same day, otherwise it doesn't work. Weirdly enough all the older stacking buffers remain at ph = 6,8
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