We are not interested in activity, so it doesn't matter if they lose activity. They just need to be able to be identified with antibody.
If the protein has been denatured and boiled, I would limit it to maybe 10 times before you start to see hydrolysis of the proteins.
In my case it never mattered how many times I freeze thaw my protein samples for western blots. I could constantly get my proteins detected.
Hello. I think it depends on the freezing temperature. Personally I freeze in -20 degrees and I had no problems in detecting till now. Probably if you will use the same samples for many times over 5 times you can have more difficulties detecting proteins with low size.
Hello. Like activity, reactivity to an antibody requires a certain intact structure. You will have to test the resilience of that certain structure to freeze/thaw cycles of that certain Protein to know for sure how stable it is, if no one did this before for you.
I agree with the above comments, if they are bolied and denatured (with LB buffer), it does not matter how many times you freeze and thaw your samples. I store them in -20ºC and they are complete fines after several months.
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