Ive been having a weird problem with my cell lysis lately. I lyse my cells, I quantify my proteins and load around 50ug sample on 8% gel and after transfer I stain my nitrocellulose membrane with Ponceau.... I don't see any bands on my membrane and I reveal the same membrane and see no band...
I use the following lysis buffer
Tris - 50mM
NaCl - 100mM
EDTA - 5mM
NP40 - 1%
I used regular HeLa cells (75cm2 flask) and scrape them in 500ul LB (+Protease inhibitors) after letting them on ice for 20 min. then I cfg the samples at 10,000 rpm for 10 min.
I collect the supernatant and quantify using Bradfords rgt. I generally get an OD of 0.1 - 0.2 which seems ok. then I approximately load 50ug protein sample on an acrylamide gel. after the transfer I see the ladder clearly on the membrane (which means my transfer was alright) and then when I stain the membrane using ponceau, I see nothing. I reveal the membrane anyways and I see noting.
I don't understand where my proteins went missing!!!!!!
There is no problem in running and transfer coz I loaded just the cell media and I see bands clearly upon transfer and then when I stain the gel with coomassie I see nothing in the gel as well.
So, I guess the problem is somewhere in the lysing step.