Immonuprecipitation of membrane proteins, is it possible? - (Nov/21/2007 )
I would like to know whether it is possible to immunoprecipitate a membrane protein. Actually, the protein I am studying could interact with a transmembrane protein, so I wanted to know if you have any good protocol to do so. Would immunoprecipitation be a good first approach to find out which transmembrane protein anchors my protein of interest to the plasma membrane? I have tried immunoprecipitating using the following buffer to resuspend purified membranes from the cells I am using:
50mM Tris-HCl pH 7.4
1mM EDTA pH 8.0
1% NP40 (or 1% Triton X)
Protease inhibitor cocktails
Unfortunately, I have not had any positive results so far. Would you suggest otherwise other methods to find out the interaction partners of my protein anchored through a transmembrane protein? I would be really grateful if anyone gave me some hints, I am stuck at the moment. Thank you all.
Membrane proteins can be IPed fine. Just see all the paper out there with membrane proteins, such as receptors or SNAREs... IP is one of the most common method they use.
You don't know which membrane protein bind your protein and now you are searching for it? As I understood, you seem to isolate the membrane part, solubilize it, then IP your protein? How are you going to see which protein interact with it? MS?
After solubilize the membrane proteins, what did you do? Spin down and use the supernatant for IP?
You said unsuccesful, meaning? Cannot pull-down your protein (the cytosolic one)? Cannot see any difference compared to the control? Cannot isolate any membrane protein?
You may not need the EDTA. Try without it and see if it have any effect.
You may want to try without detergent also. But in this case, remember to wash very carefully, profusely to lower the unspecific. Control is a must, of course. Do NOT wash using column. Membrane with not be washed away that way, and you will not see any difference. wash the beads (in a tube), spin down at LOW speed (which will NOT precipitate down your membrane, ONLY the beads), then remove the supernatant by pipetting.