I have problems with the lysing buffer for the proteins extraction for western blot. I’m using RIPA buffer (1 ml Tris-HCl 1M pH7.4, 7,5 ml NaCl 1M, 2.5 ml EDTA 0.1M, 750 ml NP40, 250 ml Na3VO4 200mM, H2O 38 ml + at the moment of use 5 ml/ml Leupeptin 1mg/ml, 8 ml/ml PMSF 100mM, 1 ml/ml Aprotinin 1 mg/ml): I add it to the dry frozen cells pellet (normally osteosarcoma cells), I mix well, leave it in ice for 30 minutes, spin at 12000 rpm for 10 minutes and then I collect the supernatant. The problem is that I always have a very small protein concentration, about 200-500 mg/ml of proteins (which is too few concentrated for the western blot), whether I’m using 50 x 10^6 in 300 ml of buffer or 5 x 10^6 in 300 ml of buffer, or 50 x 10^6 in 50 ml of buffer. It seems like it’s not possible to obtain more concentrated proteins!! Do you have any suggestion about how to increase the protein concentration?
I didn't find the right solution from the Internet.