I have tried to biotinylate some Fab fragments but I am afraid the Biotinylation is happening also at the recognition site, thus impeding the binding of my target peptides.
I would like to have some advices, suggestions, how to deal with this problem.
First do you have evidence that yoyr Fab is active in the absence of biotinylation (detection with a secondary anti-H+L antibody)? If yes, why dont' you change your biotin (most likely reacting with aminogroup) for one reacting with sulfhydryl or carboxyl groups or tyrosine or histidine?
You also may want to check if you can buy the FAB fragments pre-biotynlated. If not, as Denis pointed out, if you can't target other nucleophiles, you are in a better position than targeting amines.
You can label it with a biotin derivative reacting with the glycans on hCG.
Ulf-Håkan
I agree with Denis. You can try something. I recommend you mark the biotinylated whole antibody with high molar ratio biotin: Ab, prior to its fragmentation. Then check that continues to recognize the peptides. If recognized, then proceeds to fragment and get the antibody Fab fragments and verify again that you still recognize the peptides and still retain some residual biotin.
If it works you should not choose an alternative conjugation with other different reactive groups.
I use a biotin/ antibody ratio 4/1 (Mol to mol). Using this ratio is very unlikely that you see any alteration in antigen recognition. A ratio higher tan that (i.e. 6/1) frequently gives problems
First of all I would like to thank all of You for these helpful answers.
I discovered too late that my Hybridoma cell supernatant contained 5% FCS so I´m dealing with my target AB plus bovine antibodies. This I suppose is the reason I got strange results with my Fab fragments.
While waiting for Protein L to eliminate these uninvited guests, I made an experiment using a purified antibody we had in store here, and the biotinylated Fab fragments gave a fair result in WB and for IP. So I´m pretty satisfied at the moment.
Nonetheless I suppose I will be reducing the ratio Biotin/Fab and also doing some of the check experiments proposed, like biotinylation of the intact antibody and assessment of the Fab-activity without biotinylation.
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