Some possibilities are possible:

A: Your membrane protein isn't well expressed in your gram positive bacteria in which case you should try to increase the expression of your protein with inducers compared to uninduced bacteria. If expression is inefficient but still happening then harvest more cells.

B: Your cell lysis causes your dying bacterium to freak out and release proteases which are destroying your protein. This always happens. Thus you should be using a protease inhibitor in your lysis buffer. An efficient protease inhibitor is phenylmethyl sulfonyl fluoride (PSMF). PMSF inhibits the action of proteases. Stock PMSF solutions are soluble at 200 mM in isopropanol and not soluble in water. In your lysis buffer, PMSF should be diluted to

Dear Adrog,

Thanks for sharing these tips and your experience with me.

Probably, I should have mentioned that my protein is not recombinant, therefore increasing its expression might be a limitation. However, I definitely can try harvesting more cells.

I have omitted that I included Roche EDTA-free complete protease inhibitor in my lysis buffer. Nevertheless, I would keep in mind the PMSF option for the next attempt.

Unfortunately, I don't have access to a French press. I would try boiling the whole cell in SDS. Is it Ok to boil it in the sample-loading buffer that I use for SDS-PAGE rigth? (SDS, glycerol, beta-mercapto, and bromophenol blue).

Finally, regarding boiling the pellet with SDS I already tried this but with urea 8M instead of SDS, sady I didn't see anything in my gel either. I will try NP40!

Thanks for all your tips!

Triana