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