lysis buffer / protein concentration

John Richard Seavitt jrseavit at artsci.wustl.edu
Wed Jul 16 19:37:22 EST 1997


> 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
> 
> 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


Same holds true for 1% NP-40 lysis buffers.

John Seavitt




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