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
