Electrophorese problem

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Electrophorese problem

Post by havery01 » Mon Nov 22, 2010 9:14 pm

You have a mixture of proteins from cells and electrophorese them in a non-denaturing agarose gel in a pH 9.0 buffer. The gel is stained with Coomassie Blue and shows four bands. Suspecting there are more than four proteins, you electrophorese another sample in the same type of gel but in a pH 7.6 buffer. You again get four bands. You are still not convinced there are only four proteins.
What would you do to resolve the question of how many proteins there are in the mixture?

Me: I know that you have to use agarose gels on native proteins instead of something like a polyacrylamide matrix with SDS or 2-ME which are used to move denatured proteins electrophoretically. Agarose gels have pores large enough to allow even the largest proteins to pass unimpeded. As for the pH of the buffer used, there was only a buffer that was basic and slightly more basic than neutral. If a protein is amphoteric, that means they have a positive and a negative charge. The charge a protein carries is also determined by the pH of the solution in which it is placed. Could it be that if the protein was placed in a acidic solution more bands would show up because the amino groups are negatively charged?

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Post by JackBean » Tue Nov 23, 2010 7:03 am

I don't think you must use agarose gel for native electrophoresis. We have used also PAGE, you just do not add SDS to it ;)

Well, they are probably aggregated in some supramolecular complexes, which could you break by addition of like 0.1 or 0.2 M NaCl, but I don't know, what would that do with the electrophoresis... :roll:

Cis or trans? That's what matters.

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