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

Artem Evdokimov AEVDOKIMOZ at cinci.rr.com
Tue Sep 4 17:41:41 EST 2001


> 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.

No, no, it's not 'even' - it should be *only* 5 months of struggling.

> Has anyone else encountered protein g sepharose dissociating
> throughout the IP process??? As you can imagine, this results in a

Yeah, once. Proteolysis was to blame.

> 1) Is this normal?
No, it is not :)

> 2) How can I stop this happening??

Dima has made probably the most sane suggestion...

> 3) Any suggestions for perhaps using a more dense acrylamide gel to
> separate the bands?
> 4) Has anyone tried using a non-denaturing acrylamide gel for
> immunoprecipitation?

Actually, the immuno-gel of olden times was 'native'. Pick up a really old
bacteriology or immunology textbook, you might get lucky. These gels were a
real REAL horror to decipher.

> 5) Has anyone got a fail-safe protocol for using Sigma protein g
> sepharose in IP?

Not me :) I doubt there's a really 'foolproof' method.

A.





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