Hi, I would like to help but in order to understand possible reasons behind all those multiple problems you have described, I need to know the exact protocol you used for co-IP;

cell lysis buffer (composition); antibody (Vendor, how much per reaction, how it was coupled to the beads; before or after interaction with the protein of interest); what kind of beads you used (Vendor, cross-linked or not); how preblocking was done, if any; washing buffer you used (composition); elution buffer (composition); PAAG you used (percentage, home made or commercial).

All that stuff matters in co-IP.

One more thing: do the multiple bands look like the reactivity from the cleaved protein to you or they are below and above the full-length product (non-specific)?

We have used a Thermo Fisher kit to co-Ip 5-HT2A and D2 receptors . Please see J Psychopharmacol. 2012 Oct;26(10):1333-47; J Biol Chem. 2013 May 31;288(22):15712-24.

It worked quite good and much better that the sepharose beads that we used in the past to Co-IP GPCRs and G-proteins.. The protocos are in the papers (I do not have conflict of interests to recommend this product).

Best,

Gonzalo Carrasco, PhD

Thank you both for your reply!

Dr. Laptenko: I use the ThermoFischer Pierce Crosslinking CoIP kit with magnetic protein G beads. I make membrane prep in the kit lysis buffer containing 1%NP40. I initially followed the kit protocol (beads+antibody crosslinking followed by antigen incubation) but it didn't work. So changed it. Now I incubate 5ug antibody (Sigma) with 500ul of 800ug protein (diluted in RIPA buffer:1%NP40, 1%Sod deoxycholate, 0.1%SDS) overnight. I used different buffer after checking different ones to find one which achieves solubilization of membrane receptors but does not break the interaction of interest. I then incubate with beads for 3-4 hours. For washing I use the same NP40 buffer which comes with the kit and then last wash with water. Elution buffer is a ph2 buffer. I use 7.5% Tris-HCl gels from Biorad. Please advise and let me know if you need more information to help me with this. I have worked on it for over a year trying numerous things from sample prep to pull down and different gels to run. We have tried a lot of antibodies and found the ones which are good for IPing and detection (different species). I now see the band of interest pretty well but like I mentioned, elution is not efficient to allow any quantification and negative control shows bands. Also the vertical streaking, we think may be at some point aggregation occurs and when we run it on gel, breakdown of those releases lower MW entities which streak down again creating problem in quantification.

Dr. Carrasco, I will go over the methods of those papers. One of the receptors of our interest is D2 receptor as well, so it should be helpful.

Just to be sure . The kit that we used is: #26149. Again, no conflict of interest.

http://www.piercenet.com/instructions/2162181.pdf

Best,

I would suggest you to make ultracentrifugation of inverted vesicles - you solubilize the membrane fraction in CHAPS or other and use it below critical Micelle Concentration (CMC) later. so you will have 50% inverted lipid vesicles that you can pellet on ultracentrifuge and examine the pellet for the protein of interest. I use to make pull-downs with such a vesicles prepared from Drosophila heads.