Recovering proteins from Western Blots

Ronald Berry Berry at biochem.unp.ac.za
Thu May 5 04:38:17 EST 1994


Hello All

I have a Western blot of a protein, which has been immunodetected and 
stained with ABTS. I want to recover the original protein intact in order to 
do an electrospray mass spec study on it. Therefore, I need to firstly strip 
off the antibody (and precipitated ABTS stain?), and then release the 
protein.

Has anyboby any thoughts on how I could accomplish this? Presumably an 
acidic wash would remove the antibody (a la immuno-affinity chromatography), 
but how will this affect the binding of protiens to the nitrocellulose 
membrane (i.e. will this strip ALL protein from the blot, or, conversely, 
will the immunological binding be released, but the antibody remain bound to 
the membrane by non-specific binding?). 

Needless to say, I have *VERY* little sample available, and so trial and 
error shots are out of the question. 

All contributions will be gratefully received.

Cheers,
Ron Berry

Department of Biochemistry
University of Natal
Pietermaritzburg
South Africa



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