I'm studying the dimerization of a high-weight protein (~350 kDa), which means that I need to be able to resolve both a 350 kDa band and a 700 kDa on a gel. I've gone through several different gel systems, each with its own drawbacks, and was looking for some advice on any possibilities that I may have overlooked. Low-percentage (4%) acrylamide gels resulted in my protein getting stuck in the wells. I've tried using hybrid agarose-acrylamide gel with 2% acrylamide and multiple different buffer systems (tris-glycine, tris-acetate, tris-borate) and each one usually winds up with smearing of the 700 kDa band, which I was hoping to avoid. Switching to a vertical agarose system winds up with even worse smearing.
Is there anything that I've overlooked? I know that I'll have to accept some compromise with such high-weight proteins, but I'd like to hopefully avoid well precipitation and smearing as much as possible.
Also, are there any recommendations on high-weight protein markers? I've used cross-linked thryoglobulin, but if possible I'd like to use a high-weight monomer instead.
Hi,
while decreasing the percentage of the gel this smearing occurs. You increase the percentage of acrylamide with 12% separating and 10% stacking you may get a clear band for 700 and 350 kda. High molecular range marker should be used.
Native gel system also be used.
If u need i send u the protocol send me ur mailing add
I may have glossed over a couple of things. The protein I'm looking at is usually found as a noncovalently-bound dimer until it's activated, at which point it dissociates into monomers. I'm looking at an alternative activation pathway, however, where the protein becomes covalently-dimerized upon activation. This means that I'm trying to compare non-covalent vs. covalent dimers, which is why up until now I've used non-reducing denaturing electrophoresis for my runs. If I used BN-PAGE, is there a way that I could dissociate the noncovalent dimers, either by subbing SDS for the detergents or heat-denaturing the proteins? I agree that BN-PAGE should give me the range and resolution that I'm looking for, I'm just worried that it won't be able to highlight the changes I'm interested in since it isn't denaturing.
Thanks for the replies so far!
Hi Andrew,
did you consider using gradient gels? There is another question opened in "Biochemistry" field, where the author (Juan Gomez Gerique) asked about the best method to separate proteins between 300 - 1000 kDa. Maybe the answers there might be of use to you as well.
I've been working with IgMs and we use Novex pre-cast gradient 3-12% Bis-Tris gels - from my experiences, you can see very nicely anything ranging from monomer (~170 kDa - or even smaller) up to hexamer (~1200 kDa) without any problems.
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