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Antibodies - Problem in raising and purifying Ab (Feb/23/2004 )

[B][COLOR=blue]I am working on 565 amino acid length protein- a transcriptional factor. The whole protein was injected to raise antibodies in rabbit.
The crude serum had lot of bands so the affinity purification of antibodies was done using 4 different regions of the protein. The affinity purified antibodies were found of concentration 0.05mg/ml. The western results using affinity purified Antibody showed specificity in over expressing cells in culture.
When western were done on mice tissues, It was lighting up a totally different protein of higher molecular weight. Please suggest something to resolve this problem.


Hmmmm, sounds strange...

The affinity purification should have taken care of unspecific binding, so I can think of only two posibilties at the moment. Either your target protein is not or very lowly expressed in the mouse tissues you look at, which would explain why you don't see a band where it should be, or in mice your protein of interest is heavily modified or even has something attatched to it like a transmembrane domain or something... Maybe the band you see and your protein belong to somekind of family of proteins, so that they share similar domains the antibody could detect...