evaluating in house conjugated dynal beads

From: Labo, Nazzarena (NIH/NCI) <labon@mail.nih.gov>
Date: Tue Sep 14 2004 - 08:45:38 EST
 

Hi

I'm working with tosyl-activated Dynal beads that I conjugated with
different Abs and proteins of interest and I'm having an hard time in
verifying the actual load on the surface of the beads.

I've tried several methods, including FACS and immunofluorescence, that are
difficult because apparently those beads are per se quite fluorescent in
every wavelength but UV. 

One of the antibody I use for coating is OKT3, and I can indeed detect with
an anti IgG2a (Pharmingen), I mean I can see a completely distinct
fluorescence peak as compared to isotype control, even tough the differences
is small. The other protein of interest are IgG1 Fc fusion proteins, and a
FLAG tagged. I tried several antibodies, (SIGMA M2 anti Flag, Jackson anti
IgG1, BD anti IgG) with no result.

Is there a special protocol I have to use? Special blocking, washing?
Special setting? 

I would greatly appreciate any suggestion on how to deal with quality
control of such in-house coated beads.

 

Thanks so much, and have a nice day! 

 

 

Nazzarena Laḅ, MD

Postdoctoral Fellow NCI/ETIB, NIH

Room 12C207, 10 Center Dr. 

Bethesda, MD 20892, USA

+1 301 594 9595

labon@mail.nih.gov
Received on Tue Sep 14 13:45:20 2004

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