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Re: kinases protein kinase antibodies



On Sun, 12 Dec 1999, Steven Pelech wrote:

> Only half a dozen of the investigators who are part of this very large
> protein kinase resource network answered my plea for the names of protein
> kinase antibodies (and their suppliers and catalogue numbers) that they
> have found either do or do not work. I know that you are all very busy out
> there, but surely such information could be very helpful in your research
> programs. 

Well the list provided by Brigette Kasper to you, was very helpful to me.
I found that she seemed to prefer New England BioLabs for pp38 rather than
perhaps Santa Cruz whom I purchase from currently.  This being the case,
should my next set of data yield the same confusing results, I will order
a new antibody from New England BioLabs.  So thank you for that
information...

Steve, I may be among many here who are just starting to look at kinase
activity.  The antibodies I use are limited at this point but they have
been predominantly purchased from Santa Cruz.

I am beginning to question this however because some antibodies are quite
specific whereas others yield a high background regardless of wash time
etc during western blot analysis.


I have personally found that about a third of the commercial
> antibodies that I have tested DO NOT WORK OR BLOT VERY POORLY on the
> kinases that they are meant to target.

I think this is true--however since *I* do not have a grand array of
knowledge on antibody suppliers or there successfulness it has been a shot
in the dark for me.

 This results in a lot of wasted time
> and money. I will provide a list of recommended and not recommended
> antibodies from my personal findings and that of others to those that tell
> me about their antibody experiences.

Yes--our lab has spent more funds purchasing antibodies since August, then
any of my other type of work.

> In view of the poor response to my question about protein kinase
> antibodies, I am even more concerned about whether anyone is even
> interested in using a multi-kinase blotting service for under $2000. where
> about 100 different protein kinases could be screened by immunoblotting for
> their expression and phosphorylation states in about 2 mg of crude cell
> lysates from a control and an experimental sample (e.g. mitogen-, stress-
> or drug-treated cells, or healthy and diseased tissue biopsies). You send
> me the samples, I do the Western analysis and provide you with quantitative
> information from densitometric scans of the immunoblots as well as copies
> of the scans. This could allow you to focus on those kinases that are the
> most markedly affected in your favourite model system. Most of you will
> appreciate that the set up costs for such as analysis would be over
> $25,000. to track about 100 kinases. I believe that this is much more
> useful information than can be derived from gene expression arrays for
> protein kinases.
> 
> If you think that such a multi-kinase blotting service would be useful,
> please let me know (I will not follow up with further information about the
> service in the future unless you request it in your e-mail reply). If you
> don't think that such a service would be helpful for you, please also let
> me know. A simple "YEA" or "NAY" e-mail reply for those of you who are
> really busy would be most helpful.

I think your efforts will most likely be rewarded however, I think at this
point in time with my lab, we would most likely NOT pay $2K in order to
survey samples.  However...when grant belts loosen that would be an
interesting investment to derive mass quantities of data at one fell
swoop.


these are the antibodies I have used from Santa Cruz that I would
actually recommend:
p-ERK
p38
p56

The rest give me irregular results and I am working on finding answers as
to why?

> Thank you in advance from Steven Pelech.

Thank you for trying to make it easier for those of us just starting out.

> 

Ecclesiastes 3:1  To every thing there is a season, and a time to every
purpose under the heaven

******************************************************************************
Jeannette Corral-Research Scientist
University of Washington
School of Public Medicine
Department of Environmental Health-- Toxicology
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Phone: 206/685-1938
Fax: 206/685-4696