Antiphospho-Ser/Thr antibodies

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steven.adamson
steven.adamson's picture
Antiphospho-Ser/Thr antibodies

Hello all,
I've been working with two-dimensional IEF/SDS-PAGE to determine the pI range of my plant phospho-protein. I plan to soon buy Antiphospho-Ser/Thr and Antiphospho-Tyr in order to determine type of amino acid that is phosphophorylated in vivo.
Is there anyone who is familiar enough with the following companies to aid me in distinguishing which one has the best product?

A. Cell Signaling Technologies
B. Upstate Cell Signaling solutions
C. Sigma
D. Other Manufacturers

Thanks for the input.
Steve Adamson

nin1318
nin1318's picture
several people in my lab have

several people in my lab have used the phospho-ser/thr from upstate on 1D blots and have had pretty good results...i have never personally used it, but from what i saw the only problem was sometimes having a high background. i will ask a colleague and post more info.

tstuart1
tstuart1's picture
I've have used the phospho

I've have used the phospho-specific antibodies from Cell Signaling with great success (MAPKs). I have also used Upstate products with mixed success. I would rate Cell Signaling the best source, as they perform the most qc on their products. I stay away from big distributors like Sigma, VWR, Fischer, ect. for primary antibodies, but they are fine for secondaries. Rule of thumb is that every antibody is different, and you have to pick the company that produces the best antibody for that specific antigen. For example, I have not found many good antibodies from Santa Cruze, but a few are very good. This makes choosing a company difficult and our lab goes through a trial&error phase each time we begin a new project or use a new antibody. Hope this was of some help.

Tom

SanDiablo
SanDiablo's picture
I never had much luck with p

I never had much luck with p-ser/thr or p-tyr antibodies, though I have had considerable success with phospho-specific antibodies against particular proteins. Tom is correct in stating that you must evaluate different antibodies from different suppliers, as there seems to be a considerable amount of inconsistency in performance from one lab to another (I have postulated on the causes being everything from variable magnetic pull, to atmospheric weather patterns, and have been know to employ voodoo in the lab rom time to time!)

I preferred Upstate and Calbiochem, and warn to stay away from Santa Cruz whenever possible. Santa Cruz frequently raises antibodies against peptides, then QC's them against the same peptides, which frequently have little to do with the epitope available in the native protein.

Some people I know get around the high background issues of anti p-ser/thr or p-tyr by using them for IP, then probing the western with a target-specific antibody. This way, you can still determine which type of phosphorylation you have while minimizing the background on the blot.

Kuo-Ting Chang
Kuo-Ting Chang's picture
steven.adamson wrote:Hello

steven.adamson wrote:

Hello all,
I've been working with two-dimensional IEF/SDS-PAGE to determine the pI range of my plant phospho-protein. I plan to soon buy Antiphospho-Ser/Thr and Antiphospho-Tyr in order to determine type of amino acid that is phosphophorylated in vivo.
Is there anyone who is familiar enough with the following companies to aid me in distinguishing which one has the best product?

A. Cell Signaling Technologies
B. Upstate Cell Signaling solutions
C. Sigma
D. Other Manufacturers

Thanks for the input.
Steve Adamson

Hi, Dear

I have quite a lot of experiments with anti-phospho Abs (tyrosine/serine/threonine) that apply on western bloting (1-D). Cell Signaling Technologies is the first choice, and Upstate is the second (storage and reusage are not so good).

It is very important that sample lysate preparation must be treated with phosphotatase inhibitor (sodium orthovanadate) to protect your phospho-proteins. Details of sodium orthovanadate preparation can be found in Upstate website.

I holp the imfomation will be useful for you.

In 2-D analysis, I have no idea that does sodium orthovanadate added in lysis buffer will affect the first dimension (IEF) results or not.

Anyone who has the experiment may share it with all of us.

Good luck

jongleur
jongleur's picture
SanDiablo wrote:I never had

SanDiablo wrote:

I never had much luck with p-ser/thr or p-tyr antibodies, though I have had considerable success with phospho-specific antibodies against particular proteins. Tom is correct in stating that you must evaluate different antibodies from different suppliers, as there seems to be a considerable amount of inconsistency in performance from one lab to another (I have postulated on the causes being everything from variable magnetic pull, to atmospheric weather patterns, and have been know to employ voodoo in the lab rom time to time!) I preferred Upstate and Calbiochem, and warn to stay away from Santa Cruz whenever possible. Santa Cruz frequently raises antibodies against peptides, then QC's them against the same peptides, which frequently have little to do with the epitope available in the native protein. Some people I know get around the high background issues of anti p-ser/thr or p-tyr by using them for IP, then probing the western with a target-specific antibody. This way, you can still determine which type of phosphorylation you have while minimizing the background on the blot.

 
Dear SanDiablo:
          I was wondering what's the advantage of "IP with p-ser/thr/tyr antibody and then WB with target-specific antibody" than "IP with target-specific antibody first then WB with p-ser/thr/tyr antibody". Will the 2nd method also give a clean background?
        Thanks.