On 6/25/02 Corazon Bucana wrote:
"Recently some questions came up on how best to validate the antibodies that
we are using. I would appreciate any information as to how other people do
this sort of thing. My strategy is to do a competition experiment but
sometimes that may not be feasible especially when dealing with antibodies
to phosphorylated molecules. Any comments or suggestions would be greatly
appreciated."

Cell Signaling Technology uses a variety of techniques to validate the
staining of our products, including phospho-specific antibodies. We profile
signaling pathways in stimulated or otherwise treated cell-lines that are
fixed, processed and paraffin-embedded. We will also look at the effect of
inhibitors on downstream signaling. Prior to fixing the cells an aliquot is
removed for lysate preparation and a western blot is run. The blot result is
compared with the IHC staining. Typically when we see specific inhibition of
a protein and its downstream targets by western, we will see the same with
the paraffin-embedded cell pellets. For example, using the LNCaP cell line,
we see excellent Phospho-AKT staining in the membrane of the untreated
cells, and that membrane staining is eliminated upon treatment with
LY294002, as is the staining seen with Phospho-GSK3. 
Another approach is to preincubate the antibodies with antigen-specific or
control peptides prior to incubation on the slide. Peptide blocking is also
performed in conjunction with a western blot for verification. 
A third approach we use for the phospho-specific antibodies is to pre-treat
the slide with phosphatase prior to incubation with the primary antibody.
This works very well and nicely demonstrates phospho-specificity. This can
also be done in conjunction with western blotting. 
Perhaps any single approach is insufficient; however, when performed in
combination we feel there is ample evidence for the specificity of the
antibodies. 
I'm always interested to hear others' opinions on this! 
Katie




Katie Crosby
Manager, Immunohistochemistry
Cell Signaling Technology
166B Cummings Center
Beverly, MA  01915

Phone: 978-867-2352
Fax:  978-867-2402
E-mail:  kcrosby@cellsignal.com