Dear Olga,

I agree with the comments made above, there are a few things to take into consideration for your IP experiment :

As your protein is a membrane receptor, is the lysis buffer used strong enough to extract your receptor properly, i.e., can you detect it by WB on total cell lysates ?

I think if the antibody works for flow cytometry, it should recognize your protein during the IP if the receptor is kept in a non-denatured form. Therefore, your lysis buffer might be too harsh and your receptor denaturated and not be recognized by your antibody during the IP anymore. 

Similar to Dennis, I typically use 200 ug of protein in 200 ul of lysis buffer supplemented with protease and phosphatase inhibitor with 2 ug of antibody, overnight at 4C on rotation. The day after, I add 50 ul of protein A magnetic beads  for 2 hours, again at 4C on rotation. Make sure to wash your complexes with the same lysis buffer you used for the extraction, otherwise, you might loose the Ab-Ag interaction.

Best,

Damien

The concentration is very important for a successful immunoprecipitation. If the antibody is not validated for IP and the company gives you no recommended concentration or dilution, I use 10ug/ml antibody per sample. But most antibodies that are only for flow cytometry and immunohistochemistry have a too low concentration, so that you would need way too much to get the sufficient amount of antibody. Also the amount of protein and the volume play a role. You need as much as possible of your protein of interest in your sample and the volume has to be high enough that the antibody has "space". I use a volume of 500ul and the total amount of total protein in my samples is usually between 100 and 150ug. 

I recommend you to buy an antibody that has a minimum concentration of 0.5 mg/ml. I doesn't need to be validated for IP. 

Sadly enough, far too many antibodies do not even work properly for the application for which they supposedly are validated!

Bradbury, A. R., and Plückthun, A. (2015). Getting to reproducible antibodies: the rationale for sequenced recombinant characterized reagents. Protein Eng. Des. Sel. 28, 303-305. http://www.bioc.uzh.ch/plueckthun/index.php?pid=3-1-38300

If an antibody works for flow cytometry, it is recognizing an epitope accessible in the folded protein and ought to work in pulldown, unless you denature your protein in solubilization. How do you immobilize the antibody for pulldown- Protein A?

Appart from the aspects mentioned by Dennis, other problems to test are whether your protein gets properly solubilized and is actually present in the supernatant, and whether the interaction between your protein and the antibody is sensitive to the detergent used for solubilization.

Dear Olga,

I agree with the comments made above, there are a few things to take into consideration for your IP experiment :

As your protein is a membrane receptor, is the lysis buffer used strong enough to extract your receptor properly, i.e., can you detect it by WB on total cell lysates ?

I think if the antibody works for flow cytometry, it should recognize your protein during the IP if the receptor is kept in a non-denatured form. Therefore, your lysis buffer might be too harsh and your receptor denaturated and not be recognized by your antibody during the IP anymore. 

Similar to Dennis, I typically use 200 ug of protein in 200 ul of lysis buffer supplemented with protease and phosphatase inhibitor with 2 ug of antibody, overnight at 4C on rotation. The day after, I add 50 ul of protein A magnetic beads  for 2 hours, again at 4C on rotation. Make sure to wash your complexes with the same lysis buffer you used for the extraction, otherwise, you might loose the Ab-Ag interaction.

Best,

Damien

Dear Olga,

I agree with the comments made above. However you should also consider the nature of the membrane protein you are trying to immunoprecipitate. (e.g. one or more trasmembrane domains). I would suggest to play with detergents (CHAPS, Sodium deoxycholate, BRJ 96 etc.) in order to identify the one able to extract your protein preserving the epitope recognized by the antigen. I would also suggest to try the ab in WB directly on the cell lysate (with the different detergents) before trying the IP experiment.

Best,

Piero