immunolocalization

Sharon Barr barrs01 at DOC.MSSM.EDU
Mon Jul 20 15:16:51 EST 1998


I am immunostaining paraformaledhyde-fixed cells for subcellular
localization.  I have tried two different antibodies, an anti-T7 peptide
(Novagen)and an anti-HIS (Santa Cruz)to light up an epitope tag.  Both
antibodies are giving me a significant nuclear artifact.  That is, the
nuclei stain very brightly in every cell, not just the ones transfected to
express the epitope-tagged protein.  I have tried different washing
conditions, different blocking solutions, and different concentrations of
primary and secondary antibody.  I know it's not a secondary antibody
problem.  Any suggestions?  I realize this is not technically a fluorescent
protein problem, but I thought this group was the most likely to have an
answer. I'd appreciate any advice or protocols!
Sincerely,
Sharon

Sharon Barr
The Johnson Lab
Mount Sinai Medical Center
1 Gustave Levy Place
Box 1194
New York, NY 10029
212-241-7510
 





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