Protocol for Fluorescently Labeling Antibody Wanted

Albert Smith smith at
Sat Aug 6 09:58:19 EST 1994

I want to directly conjugate some rabbit polyclonal antisera with
rhodamine and/or fluorescein.  I have gotten the appropriate succinimidyl
esters from Molecular Probes and would like some insight into methods that
I can use to label my antisera.

In a first attempt I tried to combine fluorescent labeling with affinity
purification.  I generated a protocol similar to one used for affinity
purification/biotinylation of antibodies described in BioTechniques
13:546-8.  But I didn't end up with labeled antibody.  I know that my
affinity purification protocol alone works, so the problem is with the
fluorescent dye coupling.  

Can anyone provide an appropriate protocol for fluorescently labeling an
antibody.  I would be interested in protocols that combine affinity
purification with labeling as well as protocols for solely labeling the

As an aside, would a succinimidyl ester be reactive with nitrocellulose? 
The biotinylation protocol mentioned above used a combination of
nitrocellulose and a succinimidyl ester and ended up with biotinylated
antibody.  For that reason I attempted my labeling with a rhodamine
succimidyl ester and protein immobilized on nitrocellulose.  The end
product of my conjugation was a very red piece of nitrocellulose, which
makes me wonder if I had labeled the filter better than the protein.  Does
this theory hold any water?  I am not a chemist by any stretch of the
imagination, so I ask this question out of complete naivete.

Thanks for any information,

smith at

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