lysis buffer / protein concentration

[Kelly R. Pitts]

In article <33CA5A0C.2991 at uni-konstanz.de>,
Mark.Meininghaus at uni-konstanz.de (Mark Meininghaus) wrote:

> i have the problem that i want to mesure the protein concentration of a
> whole cell lysate of different human cell lines to compare the protein
> expression after transient transfection of plasmids with my protein of
> interest. the lysis buffers i know, all interfere with the assay i used:
> 
> bradford and smith (BCA) dont like detergents
> lowry doesnt like reducing agents
> 
> my question:
> does anybody know a good lysis buffer that i can use for making whole
> cell lysates and that doesnt interfere with the standard protein assays
> (bradford or BCA)?
> 
> thanks for help
> 
> mark

We've been doing the same thing is our lab.  I take a confluent 100mm dish
of cells (actually these are stables, not transients) and dissolve the
membranes with 700Ñ1000 microliters 1x RIPA buffer (from Harlow and Lane)
with protease inhibitors.  After a ten minute incubation/lysis on ice I
collect the lysate and do a BCA protein assay (Pierce).  The
concentrations of detergent in the 1x RIPA are within the range Pierce
says is OK.  I haven't had any problems with getting good readings.

Kelly Ray Pitts
Mayo Clinic
pitts.kelly at mayo.edu