I did a one dimensional SDS - PAGE. Stacking gel is 5% and Separating Gel is 12%,however, they didn't polimerize. I tried to use progressive concentration of Acrylamide from 12% to 36%, and at 36% they polimerized but not totally. They were still sticky and dissolved away when they were put in the water or staining solution.
Is gel/Acrylamide broken down?
Thank you so much
Hi Phuc
yes that definitely sounds like a polymerisation problem. How old are your acrylamide bis acrylamide TEMED and ammonium per sulphate. The latter 2 are responsible for cross linking your acrylamide with your bis acrylamide to form your set acrylamide lattice. APS unless made from fresh or made and then frozen can break down culminating in incomplete polymerisation and only partial setting. TEMED over time at room temp also breaks down and old TEMED has a charartetistically pungent fishy (amine) smell. Similarly acrylamide breaks down onto acrylic acid and ammonia. Thus when making acrylamide gels the main reason they do not set as a one off is that a simple error like failure to add APS and or TEMED has occurred. We have all made this mistake including me. Alternatively if this is an ongoing problem I would buy in fresh TEMED APS and acrylamide particularly if any or all of these reagents are 1 year or more old
Dear PhD. Hall
I'm sorry about replying late. I don't know the reason why I had two same questions posted.
Thank you for your sharing. I also received many advices as you advised me. I also think that my acrylamide broke down. I will freshly prepare APS solution and buy a new acrylamide mix solution.
I think I got many mistakes because I just bought acrylamide, APS, TEMED,...1 month ago =.=.
If I cast gel with only acrylamide, not bis-acrylamide, is it ok, sir?
(this is a link to a same question)
https://www.researchgate.net/post/Could_someone_help_me_answer_a_question_about_SDS-PAGE
Hello Phuc
normally acrylamide as it is called is acrylamide: bis. People just refer to it as acrylamide. If you look on your bottle you should see numbers like 19:1 or 29:1; or something like that. Have a look. This refers to the molar ratio of linear acrylamide molecules to bis acrylamide. The bis is the cross linking reagent. This is what converts your linear acrylamide polymer to a cross linked acrylamide matrix which causes it to set. The polymerisation of your acrylamide bis solution is catalysed by the APS and TEMED. The ratio of acrylamide to bis determines the effective pore size in the polymerised or set matrix and therefore whether large or small molecules are more effectively resolved. Generally speaking 19:1 acrylamide bis which is best for Protein work whereas 29:1 which has larger pores is preferable for DNA/ RNA work
Normally when gels do not polymerise or set it usually means that the reagents are too old although at 1 month they should be fine; you have forgotten to add either APS or TEMED although I doubt you would keep making that mistake or one of your reagents is wrong; as you say the acrylamide ? Keep in mind as well that you need either to make up 10% APS fresh every time - if you are having polymerisation problems I would definitely recommend this - or store the solution frozen; do not freeze thaw more than about 3 times and keep just for 1 month. For immediate use if you are running a lot of gels in 1 week 10% APS is ok in the fridge for about 1 week although personally I only ever keep until the next day. Good luck !!
See below for the sort of solution you should be using. All life sciences companies will sell this
https://www.lifetechnologies.com/order/catalog/product/AM9022
Dear PhD. Hall
Thank you so much for your careful advice.
I booked a acrylamide solution as you recommended, but it's from Sigma. I will get it on next 2 months.
What do you think when I need 35% acrylamide to polymerization, but it is dissolved when i put it into water? is it abnormal, sir?
Hi Phuc
not sure I understand your question, but I think you are asking should a 35% solution of acrylamide Bis dissolve in water; The answer is yes. Many years ago we routinely made up a 40% solution from powder. However, acrylamide powder is toxic so it has become normal to by the type of solution I recommended
Doesn't matter that is is from Sigma: This liquid acrylamide/bis acrylamide solution is sold by Fisher Sigma Gibco life sciences VWR. Lots of companies !!
Dear PhD. Hall
Thank you so much for your careful advice. I will try again when I get acrylamide solution.
Best Regards
I am very happy to talk with you, sir. Thank you.
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