intracellular FACS staining - HELP!
Kiley R. Prilliman
tiey at aol.comjunkbloc
Wed Feb 16 03:50:46 EST 2000
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 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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