5 Months of Immunoprecipitation Woes - Disassembly of Protein-G Sepharose

Ian A. York iayork at panix.com
Tue Sep 4 08:05:40 EST 2001


In article <350b1ac.0109032137.2a7dabaa at posting.google.com>,
Melissa Greeve <gliftor at hotmail.com> wrote:
>
>Has anyone else encountered protein g sepharose dissociating
>throughout the IP process??? As you can imagine, this results in a
>huge band at 20kD, representing protein G, that binds and fluoresces

This should not happen.  I've never seen it, in hundreds (thousands? I 
haven't kept track) of IPs.  I've also never used Sigma protein A/G, and I 
suspect that's related.  Try a different source.  It may be more 
expensive, but it can't possibly be mroe expensive than 5 months of your 
time.

For some reason, I've always found protein G less reliable (more 
background, worse binding) than protein A.  Are you sure you need the 
protein G?  There are also modified protein G versions, with lower 
backgrounds, and I have a vague idea that there might be a different 
protein with a similar antibody binding spectrum, too.  But if you can go 
with protein A, do so.  

If you have lots of the primary antibody, consider coupling it directly to 
sepharose.  That way you'll get rid of not only the protein G band, but 
the antibody bands as well.  The protocol is straightforward--I think it's 
Pierce that sells a kit, probably several other companies do as well.

You're doing IP-westerns.  Those, basically, suck.  They're much less 
reliable than straight IPs or straight westerns: you have all the errors 
of each procedure, and they amplify each other.  Try to avoid them if 
possible.  Can you metabolically radiolabel your sample and run a 
straight IP?

If not, you might try a different approach to the IP-western.  You can 
take your lysate before IPing, and biotinylate it (or iodinate it; 
biotinylation works, and you avoid the hassle of dealing with I-125).  
Then quench the biotin, IP, transfer to nitrocellulose, and probe with 
HRP-streptavidin and use one of the chemiluminscent kits to develop.  
Your antibodies and your protein G won't be biotinylated, so the only 
things that will light up will be the proteins that were actually in your 
lysate.  This is quite a sensitive technique--more than IP-western with 
antibody, in my hands.

But the first thing to do is throw away your Sigma protein G and get some 
good-quality stuff.

Good luck.

Ian 

-- 
    Ian York   (iayork at panix.com)  <http://www.panix.com/~iayork/>
    "-but as he was a York, I am rather inclined to suppose him a
     very respectable Man." -Jane Austen, The History of England




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