> It is far easier to use a sandwich technique, if your primary antibody is
> unconjugated. This can be successfully used even with 2 and 3 color stain-
> ing.
> Trying to conjugate an antibody is time consuming and expensive if you are
> buying the unlabeled antibody. You need sufficient antibody to start - at
> least 100 ug and 1 mg would be better. After conjugating you must remove
> excess FITC, unconjugated and over conjugated antibody.
>
> If you need protocols for sandwich techniques, I'd be happy to send them.
>
> Best regards,
> Tony Bakke
> bakkea@ohsu.edu
Is this always a better idea? I did an experiment a while back in
which I used indirect ("sandwich") staining with an antibody against MHC
class I. By the time I got around to the analysis, the light scatter prop-
erties of the cells that were positive for my antibody were all out of whack,
as if I had stimulated them. It reminded me of a technique I once used for
stimulating T cells, in which you added anti-CD3-biotin and then streptavidin.
So, if cross-linking the antigen in question would stimulate your
cells, perhaps you want to use direct-labeled antibodies. Comments?
- John Ladasky
ladasky@leland.stanford.edu
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