I am facing a serious problem with SDS-PAGE, that too all of a sudden, my gel gets sticky and cannot be rmoved from the casting plate for further processing. I do take care about the requirements, pH, etc. I am unable to find out what is going wrong, can anyone help me out? I follow Biorad protocol.
Sometimes this happens if there is an issue with cross-linking (or lack of cross-linking).
I have encountered terrible problems like this - it took 3 days of remaking sticky gels and cleaning plates to correct. When I ran out of what we refer to as the "acrylamide" stock solution in the lab we re-ordered this - but our purchasing clerk made the mistake of ordering pure 40% acrylamide instead of the 37.5% acrylamide-2.5% bis-acrylamide stock solution. Consequently there was no cross-linker in the mixture. Everything else was replaced - buffer, SDS, APS, TEMED - and still the resolving gel had the consistency of thick toffee, even if I put in an excess of APS and TEMED.
Check your acrylamide / bisacrylamide stocks, and make sure APS and TEMED are fresh. Good luck!
Hii Basu, may be your gel are not polymerizing properly, the other important we should use fresh APS. this link may answer your question --> http://www.scientistsolutions.com/forum/protein-detection-western-blots-gels-ip-protein-gel-electrophoresis-and-staining/problem-0
Sometimes this happens if there is an issue with cross-linking (or lack of cross-linking).
I have encountered terrible problems like this - it took 3 days of remaking sticky gels and cleaning plates to correct. When I ran out of what we refer to as the "acrylamide" stock solution in the lab we re-ordered this - but our purchasing clerk made the mistake of ordering pure 40% acrylamide instead of the 37.5% acrylamide-2.5% bis-acrylamide stock solution. Consequently there was no cross-linker in the mixture. Everything else was replaced - buffer, SDS, APS, TEMED - and still the resolving gel had the consistency of thick toffee, even if I put in an excess of APS and TEMED.
Check your acrylamide / bisacrylamide stocks, and make sure APS and TEMED are fresh. Good luck!
Try, cleaning the plates in ethanol solution and leave it to dry for around 30min before using, this would prevent any sort of faults on unnecessary adds on the plate. Now try using rain-X, this is most preferred method, apply rain-X to the plates and rinse off the excess reagent with 70% ethanol , before use. Rain-X will act as a silicon repellent.
I think this should work.
Verify also that the acrylamide percentage is correct, and perharps there is a warming of your running system. The most probably root cause of that is your reagent stock (APS, buffer temed...)
Thank you all for your kind attention, I will take into account your suggestions and come back
Use freshly prepared, 10% APS. Nd check for any precipitation in the running buffer
After you have identifed the problem with the stickiness of your gel, you can always salinize your plates or my lab mate uses Rain-X to prevent the gel from sticking to the plates.
I would recommend you to check if your APS has gone bad. Make some fresh 10% APS and also you can use RAIN-X to prevent gels from sticking to the plates.
Dear Basu, Prepare fresh 30% acrylamide, 10% APS and check ph to Tris cl is up to the mark or not.
Clean the plates properly
Prepare the fresh APS
Mix the reagent mixture properly after adding TEMED and APS
Let the gel polymerize for 1 h at least.
Also check the pH and concentration of solutions you are using.
Hope it will help you,
Best luck with your research
I would recommend a correct homogenization of the mixture after adding fresh APS and TEMED.
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