ELISA Ab question - denatured protein and extract - If I extract denatured protein... (Nov/09/2006 )
I'm working out an ELISA for a plant protein. My capture antibody will go on my 96 well plate, I will then try to detect my protein from crude protein extract.
To make my antibody, I want to extract my his- tagged protein (from e. coli) using a denaturing protocol then send it away to get antibodies made to it.
For my ELISA - If my crude plant extract is extracted also using a denaturing protocol, my antibody (stuck to my ELISA plate) should be able to find my denatured protein - right? since I sent the denatured version to make antibodies to ????? Any problems here?
It seems fine to me as long as there is enough antigen in your crude extract, and it is not inhibited in any way from binding by other components of your extract. The only thing I am worried about is that your background may be high due to the crudeness of your extract prep.
Should be fine,
however, ELISA are not always easy to set up.