intracellular FACS staining - HELP!

Kiley R. Prilliman tiey at aol.comjunkbloc
Wed Feb 16 03:50:46 EST 2000


Hi,

I am trying to screen some transfectants expressing an intracellular protein by
FACS but have been having some problems.  I am permeabilizing the cells (mouse
endothelial cell line) with 0.25% saponin.  In the beginning, I would follow
this with a first-step (biotinylated polyclonal rabbit antisera to the antigen,
1:500, 30 min), wash, and then a second step (streptavidin-PE, 1:500, 20 min). 
However, my controls stained as brightly as anything else!  To try to lessen
this intensity, I added a step before my original first step: right after
permeabilizing the cells I would block for 30 min with 2% rabbit serum.  This
did not help at all.

has anyone else encountered similar problems, and what did you do to work
around them?  I really need to screen these transfectants!

Thanks so much for any advice; if possible,  please reply to my e-mail address
in addition to the newsgroup.

Kiley

Kiley R. Prilliman, Ph.D.
Postdoctoral Research Fellow
Division of Immune Regulation
La Jolla Institute for Allergy & Immunology

kiley at liai.org




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