Protein not penetrating the resolving part of the gel - (Jul/15/2005 )
i am trying to run a protein on an SDS PAGE gel (i use mercaptoethanol in my loading buffer or not) and although i would expect to see it at a size of 120-150 kDa, the protein does not seem to penetrate the gel and it "stops" in the top of the resolving gel.
I have also tried to run a few other proteins of various sizes up to 100 kDa and they penetrate the gel fine. I took the percentage of the gel down to 7% but it still won't penetrate.
As this is getting a bit frustrating for me now, could anyone suggest of possible solutions to the problem?
I am routinely running a 145kDa protein on 7.5% resolving gel with a 3% stacking gel and not having any problems... is this a transmembrane protien? What is your %SDS? Maybe you need to increase it.. or perhaps pour yourself a 4-30% gradient gel.
it is a transmembrane protein and i am running cell extract containing the protein of interest. I will try increasing the dtergent and i will chewch for lower percentage gel.