5 Months of Immunoprecipitation Woes - Disassembly of Protein-G Sepharose
klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Tue Sep 4 07:40:34 EST 2001
gliftor at hotmail.com (Melissa Greeve) wrote:
>Like all PhD students, I have come across one of those annoying
>'little' hitches that even 5 months of struggling can't seem to solve.
>I wonder if anyone can help me. I have been trying to get
>immunoprecipitation using protein-G sepharose to work for some time
>now, and although I have tried a couple of different buffers, multiple
>protocols, slower centrifugation, using unboiled samples and replacing
>products, it still won't bring me any joy. Along with the normal
>problems of interference from antibody heavy and light chains, due to
>the use of the same antibodies for both IP and western blotting, I
>have come across a problem that seems to be unique to me....
>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
>when the blot is incubated with any antibody type. This wouldn't be a
>problem if I was looking for proteins that don't sit near 20kD, but of
>course, 2 of the critical proteins I need to look at are about that
>1) Is this normal?
No. (Unless you mean problems some sort of problem with IP :-))
>2) How can I stop this happening??
Buy good G-protein sepharose or better yet crosslink antibodies
to the sepharose (Pierce sells "kit" for this trivial procedure).
>3) Any suggestions for perhaps using a more dense acrylamide gel to
>separate the bands?
Might work for your particular case. Start with 14% perhaps
>4) Has anyone tried using a non-denaturing acrylamide gel for
Guaranteed to be a horrible mess.
>5) Has anyone got a fail-safe protocol for using Sigma protein g
>sepharose in IP?
No such thing. IP is the most fail-prone and artifact-ridden method
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