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Co-Ip - bands in my pre-clearing controls - pre-clearing step - how to improve it? (Nov/05/2007 )

Hello hello.

I am currently immunoprecipitating my protein complex on Sepharose G.
The thing is, I get the appropiate band in the preclearing step as well.
Is my IP non-specific or does my protein have some affinity to protein G?
And what to do to ensure specificity?
The cell extract controls work just fine.

I do not have any other antibody for my protein suitable for IP and there's none available at the moment.

-Telomerase-

Your protein seems to have some affinity for protein G.
You could try to increase NaCl concentration up to 0.5M for incubation and washing steps.

-Missele-

QUOTE (Missele @ Nov 5 2007, 02:20 PM)
Your protein seems to have some affinity for protein G.
You could try to increase NaCl concentration up to 0.5M for incubation and washing steps.


Hmmm, I am currently using 50mM KCl, maybe the stringency is too low indeed.
Thanks, I am going to try this one out. Maybe not 0.5M, nevertheless higher...

-Telomerase-