No proteins after HeLa cell lysis - (Aug/21/2013 )

Hello,

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...

to elaborate,

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.

Kindly, help....

Why don't you stain a gel before transfer?

If it is a normal bradford assay, then it isn't compatible with that much NP-40 (assuming it is NP-40 used, not nonidet-P40!), you need to use 0.25% or less, which could be giving you the strange readings. 

It could also be that your kit is old and the reagent has gone off.  What do your blanks look like?

Are you doing SDS-PAGE or native PAGE? for SDS-PAGE, samples must be tretaed with SDS sample buffer (Laemmli buffer) before transfering to the gel