Use a cellulose membrane concentrator or dialysis. Those methods don't alter the function of the proteins like salting out may.

Do you have a positiv control in your Bradford assay and know for sure, that it works properly? Which cells, how many cells and how much buffer are you using? Are you trying to make a co-immunoprecipitation of endogenous proteins or do you over express your proteins of interest?

the question is do you have problem to precipitate your protein or to detect the co precipitated interactant ? in the case of direct immunoprecipitation the concentration of the protein of interest is generally not a problem (or it is because the protein is very very scarce) if you have a good antibody (maybe run the antibody-antigene reaction for longer time (overnight at 4°C with rotation). If it is the interactant then it can dissociate because the concentration is too low. I would rather work on recovering a more concentrated lysate (more cells less buffer...) than on concentrating a lysate (immunoprecipitation is already a concentration...)

Marie Caroline Demmler

Dear Marie,

I am trying to make co-IP for overexpressed proteins of interest. I used 1 ml lysis buffer for a 10 cm plate containing 70% confluency of HEK293T cells.

Didier Poncet

Dear Didier,

Thank you for your answer. Actually, I have a problem detecting the co-precipitated interacting.

I will try less buffer and more cells.

Mahsa Shirin are you sure that the protocol that you're using does work in general?

Marie Caroline Demmler Yes, I am using the protocol recommended by Invitrogen for the co-IP using magnetic beads.

OK if it is the interactant protein that you cannot detect it could be more complicated than concentrating the lysate; interactant binding can mask the antibody epitope (real concern if you use monoclonal)=try other antibodies (if you have) lower or higher concentrations of antibody. The ratio of complex (versus free protein) and/or its stability can be low: you can work on the composition of the lysis buffer (pH, salts, higher or lower nacl Mg, Ca, KCl instead of NaCl (supposed to be closer to the inside cell media...other detergent (tween, Np40)... use cross linkers .... try other methods to see the interaction or the proximity of the 2 proteins (Fret, Bret, two hybrid, split luciferase, confocal....).