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
