Co-IP - (Aug/20/2007 )

Hello everbody,
I am doing IP in particular Co-Ip.
My protocol:
a) cell harvest and lysis
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

When I've done Co-IP experiments, I've done things a little different:
before adding my beads to my cell lysis, I've also washed the beads once with buffer (plus 0.01% Tween-20)
then after incubating with the beads, I've washed it 4 times: twice with buffer plus detergent and twice with buffer without detergent.
Vortex and centrifuging the sample after each wash.
After boiling the sample, centrifuge and use the supernatant.

Also, my incubation times are also a little different: I incubate with my ab o/n and incubate with the beads for 2 hrs at RT.

Hopefully, that works. Good luck!

Yes, I would also suggest wash the beads first. Your incubating time is Ok with me though. You are sure that the interaction is real, right? Is it possible to have positive control, e.g. some known interacting parners? If the problem is really due to nonspecific binding, you need to wash more and maybe under more stringent conditions.

A few more questions, are the IPed proteins cytosolic or membrane bound? Are you using total cell lysate? If yes, when you make the lysate, which speed did you spin down the lysate (to get rid of the debris, un broken cells...) before adding in Ab? The reason I ask is that if the IPed proteins are membrane proteins (still bound with membrane even after lysis) and you prepare the lysate at speed less than 3Krpm, then when you wash the beads, the free membrane will also come down together with beads. Then when you run the gel, you will got these free membrane proteins in all samples.

I didn´t mention that I fractionate the cells in cytosol and nucleus. When I get the cytosol I sonicate (25%, 10 sec) after that I spin at 14.000 x g 90 sec and take the supernatatnt for Ip.

My protein of desire is membrane bound but it´s also located in cytosol. But the interaction partner is predominately located in the cytosol and nucleus.

I only wash twice the beads (Amersham) with 1x PBS to get rid of the ethanol in which the beads are solved.
Could it be a possibility to incuabte the beads with BSA? Maybe I can block unspecific binding sites.

Tiffy