Protocol for Fluorescently Labeling Antibody Wanted

Flip Hoedemaeker, CvF RUL/TNO, Leiden, The Netherlands sbtnfh at rulsfb.LeidenUniv.nl
Sat Aug 6 16:52:47 EST 1994


In article <smith-0608941558190001 at commons-107-node.net.yale.edu>, smith at minerva.cis.yale.edu (Albert Smith) writes:
>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
>antisera.

Hi Bert,
I've used the following protocol for fluorescent labeling of a lectin, it 
will work for antibodies as well.

 - Make a solution of 2 mg antibody in 1 ml 0.1M NaHco3 buffer pH 8.5
 - Add a solution of 1 mg Fluo.succ.ester in 850 ul DMSO
 - Shake for 1h at room temp
 - Remove free label on a small desalting column
 - (Lyophilize your protein). 

You can affinity purify your antibodies afterwards, that is the most simple
thing to do. (and you should have a beautiful visual control over the
procedure!)
Keep everything in the dark as much as possible.
Flip



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