viewing pentamers on a western blot - (Nov/17/2005 )
I ran a western blot to detect my protein which has both monomeric and pentameric forms. To detect the pentamers, I did not use BME in my sample loading buffer, and did not boil the samples prior to loading. I got bands which correspond to the pentamer size but also got a band which was running lower than the monomer. In the boiled sample, I had BME in the buffer, and those samples showed a perfect monomer, with no pentamers and no lower band.
My question is that when you do not denature the protein by boiling or using BME, can you still say what the molecular size of the bands are?
I think the answer is No.... Someone else should confirm...
So my monomer could run lower than the expected size then I suppose. Since I see a band around the size of the pentamer, can I say that pentamarization seems to have happened?
So the monomer runs lower than expected but the pentamer is the right size? I think this is possible, but how can you know it is a pentamer and not less (dimer etc.)?? Do you see intermediate products with this?? If there are only two forms I think I would agree with your assessment, but I really am not an expert in this and someone else should confirm....