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For the protein in your cells to activate T cells, it would either be secreted, cell surface, or a peptide derived from a protein that is processed and presented by an MHC molecule.  From what you describe, it sounds like you think it's the third case.  I'm not sure a total a cell lysate will be processed to produce the same complex, but I suppose it's possible.  Are there publications describing this approach?   If so, the papers should give you some guidance.  Otherwise, you could try a few different lysis methods.  Perhaps simple shearing or hypotonic lysis for cytoplasmic contents, 1% NP40 for some membrane proteins, and RIPA for most stuff.  You might also consider some type of limited proteolysis.  It might take a while to develop a good method, but that would also make the work more significant.

Since, its is not very clear whether its a membrane or cytosolic protein, I would recommend using RIPA buffer to start with. You can also try using the total protein extraction reagent from thermoscientific (M-PER Mammalian Protein Extraction Reagent). It is a very good efficient reagent and is also not very harsh, so that the proteins are not destroyed. We use this reagent in our group and it works well with most kinds of proteins.

Dear Michael and Deblina - first, thank you!

Michael, I forgot to mention that my cell line is a B cell line... So, if it somehow matters, it itself can serve as an APC. Thus, for example, it's possible that one B cell secrete something and its neighboring B cell presents it, and vice versa. I'd try using for presentation DCs but also CD40-activated WT B cells.

There are quite a few papers using whole-tumor cell lysate to pulse APCs (usually DCs) and use the APCs to present antigens to T cells. It's just that I didn't find so far papers in which the lysis method was sufficiently described and in which it was clear that the lysis was meant to try and recover both membrane and cytosolic proteins. In any case, about RIPA - wouldn't the SDS in it denature my proteins? I'm not sure, but I guess it'd be better to avoid denaturation of the proteins.

Deblina - same question to you about RIPA. About the M-PER - I wonder if it should work also with membrane proteins (so, both cytosolic and membrane proteins), because ThermoFisher has also another buffer which is specific for membranes.

RIPA buffers generally have only 0.1% SDS.  This could certainly denature some proteins or interfere with protein-protein interactions, but it's not as harsh as protein sample buffer, etc.  I actually lyse cells in RIPA and do coimmunoprecipitations all the time, so some structure and interactions must be preserved.  If your protein is nuclear, it's probably the easiest way to solubilize it.  There is a hypotonic lysis method that is used to make nuclear extracts that could be used instead.  If you don't want to worry about nuclear proteins, I think you could do a 1% NP40 lysate and see just see if your assay works.  You also could try to email the PI on one of the papers you have looked at for technical advice.  Good luck!

Think it´s almost imposible to extract or identify all protein in such material. Some will remain aggregated. Jorge,

Thanks a lot Michael and Jorge!

One question that I suddenly thought of, though, and thus decided for the time being to switch to a freeze-thaw method: I use detergents in the chemical lysis option. But then, I want to "throw" those lysates on DCs; wouldn't the detergents interefere with the DCs function or even kill them?

I agree with Michael about the quality of RIPA buffers that they are not so harsh, so you can try it. Regarding the M-PER buffer from thermoscientific, we generally uses this buffer for lysis in our group, some of us have membrane proteins and others not, it actually works. If you are sure that your protein is a membrane protein then you can try the one which is specifically used for membrane protein extraction. good luck.

Thanks a lot Peter and Deblina, very interesting and useful advise.