Problems with Co-IP

[Tiffy]

Hi,
I am doing IP in particular Co-Ip.
My procedure:
a) cell (MCF7) harvest and lysis
b) 1µg polyclonal ab per 100µg protein lysate, incubation 1h @ 4°C
c) adding the beads, incubatin oN @4°C
d) washing the complex three times with Ip-Buffer (20mM Tris/Cl, pH7.5, 500mM NaCl (!), 1 mM EDTA, 1 mM EGTA, 0,1%TX-100), centrifugation 3.000 rpm 10' , after last the centrifugation I add 25µl 2x SB +5% BME, 95°C 5'
My controls are the isotype of my ab and just the beads. After the gel run & blotting I incubate with a different ab to detect a protein-protein interaction. Unfortunately I get positive signals in my controls.
What`s wrong? What shall I do? Any suggestions.
Thanks a lot!
Tiffy

[danfive]

Your negative control (the capture antibody) is producing a signal, right. Then the problem is that the detection antibody is binding to the capture antibody and any other Ig. You can (1) denature the antibody-antigen complex with reducing buffers (identify the antibody/antigen out by size). You can (2) crosslink your protein a/g beads to the capture antibodies and elute only the antigen. And if possible (3) have a Western Blot biotin-labeled primary antibody with streptavidin-HRP conjugate for detection.

BTW 500mM NaCl in the IP-buffer seems high, I usually use .14 or .15M NaCl.