How to measure total protein conc. of cultured-cell lysate?
Richard Van Frank
vanfrank at iquest.net
Thu Dec 5 12:39:39 EST 1996
In article <gjenkins-0412961822050001 at 126.96.36.199>,
gjenkins at facstaff.wisc.edu (Glenn Jenkins) wrote:
>> 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.
You don't need the phenol red in your medium. Either use a medium that does not
contain it or wash the cells to remove it. You can then use the fluorescent
assay described in Analytical Biochemistry 65, 552-555 (1975) which was
developed for cells, cell fraction, and homogenates.
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