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total protein assay before western blot - (Jul/25/2007 )

Dear all,
I am going to immunoprecipitate a protein and then western blot. I don't know if I have to determine total protein after immunoprecipitation and before western blot or not.


ok you need to quantify ll substances.
I think that you can estimate your purification by loading on a gel the crude fraction and then maybe your washes and the eluted fraction. You have to estimaite the crude protein concentration to be able to know how much proteins were submitted to purification for further requirements.
But for WB... well to see if the protein was purified, 1, 2 and 5µl of a 50µl purif for ex should do the job.
If you need to add your purified protein to an assay, you will need to quantify precisely.