Hi, I've got a problem with my Western Blot. I have a low weight protein to detect but I suspect that my primary and secondary antibodies are clumping into a large ball at a higher molar mass than my protein. I heard that polyethylene glycol coulg help with that, but I can't find any protocol. Did any of you use polyethylene protocol to break down antibodies in Western Blot?
What concentration are your primary/secondary antibodies?
What are you blocking with?
How would clumping affect detection of your protein of interest if it is lower mwt?
Why do you think your antibodies are clumping?
What is exactly the problem with your western, is it because the antibodies are picking up a band of protein that is of higher molecular weight than what you expect?
Polyethylene glycol is a molecular crowding agent. PEG promotes binding at lower concentations of antibody and antigen. It does not break down Ag-Ab complexes.
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