How to measure total protein conc. of cultured-cell lysate?

[Glenn Jenkins]

> On Wed, 04 Dec 1996 12:17:37 -0500, Mohamed BaSalamah
> <mbasal at med.unc.edu> wrote:
.......
> >I want to ensure equal loading of two different cell lines-cell lysates
> >for western analysis. That's why I want to have total protein conc.
> >
> >  The media for these cells contain phenol red.
> >  The lysis is done using 2% SDS.
> >  The protein of interest is likely to be glycosylated.


> >Mohammed BaSalamah
> >Grad. Student
> >mbasal at med.unc.edu
> Dear Mohammed
> in my opinion it is almost impossible to make a protein assay if you
> use SDS as lysis detergent-how about using some non-ionic detergent
> such as NP40 , Brij , Triton X-100, ....? You could centrifuge off the
> nuclei and afterwards do a Bradford assay using the Bio-Rad reagent
> e.g.-but this will not work with SDS, as SDS itself reacts with the
> Bradford reagent.This assay will also work for glycoproteins as the
> dye used here is adsorbed to amide bonds of the protein!
> Hope this helps-  Martin

Since when can't protein be measured in SDS????? I think you have this
backwards. The phenyl ring of detergents like triton actual interfere with
the absorbance in the 280 nM range which forces people to use a protein assay
kit that measures protein at a different wave length.

Here is my protocol for fibroblasts:

Remove media wash gently 2x with PBS.

Add lysis buffer consisting of 20 mM tris, 1 mM bME, 1 mM EDTA and 1% SDS
(About a ml for a 10 cM plate) and mix well with pipette.

Remove and clarify for 10 min at 15K

Use 10-40 ul in 1 ml of lysis buffer of OD280 determation (Blank against
lysis buffer alone)

the extinction coeff. for individual proteins varies but the general 
rule of thumb to that the average extinction coeff. of a lysate 
is 1.0 thus 1 OD is 1 mg/ml.


Glenn

-- 
Glenn H. Jenkins
Dept of Human Oncology
University of Wisconsin
Madison, WI  53792

Ph (608) 263-4084
Fx (608) 263-4226