how to reduce unspecific binding of proteins to the beads.. - IP-purification (Jun/19/2007 )

Hi there,

I am identifying binding proteins of a transfected protein with a tag by IP and MS. from silver staining, it is clear that the bait has been pulled down. However, the IP system (beads+antibody) pulled down a lot of unspecific bands from non-transfected cell lysate...

Does anyone have any idea to get rid of these unspecific binding proteins? They make a lot of trouble for MS step...

Thanks,

-yeping-

Is a pretreatment of your sample with control Ab-beads possible?

-genehunter-1-

QUOTE (genehunter-1 @ Jun 20 2007, 03:03 AM)

Is a pretreatment of your sample with control Ab-beads possible?

Or can you affinity purify your antibodies first? How about some extra wash steps, wash steps in low-concentration detergents or some different salt concentrations?

-ScottC-

Thank you both for the suggestions.

I am thinking the control Ab treatment, I still don't know whether it's possible or not..

I tried different salt concentration, background is different after washing, but still a lot... I don't know how high concentration of salt I can go, 500mM?

I have another question, does KCl or NaCl matter? I mean in one protocol, salt means KCl but the other one uses NaCl.. (I used KCl protocol.)

-yeping-