[Histonet] Demonstration of antigen on cell surface - question?

Luis Chiriboga Luis.Chiriboga <@t> med.nyu.edu
Mon Jun 20 17:53:03 CDT 2005

I think a bit more info is needed but here's a few suggestions....
If your working in frozen or formalin fixed tissue, serially dilute both
primary and secondary antibodies independently to arrive at your best signal
to noise.
If you are performing antigen retrieval (formalin fixed), run a time series
of different HEIR times or various concentrations of enzyme.
This should help you improve the S/N to an extent that allows easier
If your working with a novel protein...
If you have access to cell culture, perform cell fractionation and check
localization via western blot.
Have you checked in the literature? for similar class or family of protein
and it's localization?
If you have/know the target protein/antigen check for protein localization
signal (NCBI-protein database & genbank), or protein sequence homology to
other known proteins (NCBI-Blast)

Luis Chiriboga Ph.D.
NYU Cancer Institute and
Bellevue Hospital Center
New York University School Of Medicine
Department Of Pathology 4W27
462 First Avenue
New York, N.Y. 10016
W(212) 562-4667.
F(212) 263-2041

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu]On Behalf Of GT Hebert
Sent: Monday, June 20, 2005 3:49 PM
To: Histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Demonstration of antigen on cell surface - question?


How would one go about determining if your specific antigen (say for IHC) is
located on the cell surface?  Right now it seems that the antigen is
staining everywhere. (I am running a sheep anti-mouse 'antigen' and using a
rabbit anti-sheep biotin conjugated with ABC + DAB for detection.  (My
negative is normal sheep IgG -- is negative).  Is there a way through immuno
or other that I can specifically say that the antigen is present on the cell
surface?  Are there general cell surface markers that I can use along with
my primary?  Thank you all.

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