I am working on protein quantification of yeast cells using lysis buffer (2% SDS, 2% beta- marcaptoethanol, 0.05M EDTA and 0.1M NaOH). The blank lysis buffer is also giving colour with the Lowry assay. Any possible suggestions would be appreciated. Thanks
The Lowry assay is fairly compatible with SDS, but not with so much mercaptoethanol (2% is about 250 mM) or other reducing agents.
Here is a compatibility table for some protein assays:
https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0068-Protein-assay-compatibility.pdf
Leave out the mercaptoethanol if you can. If not, your best bet is probably the 660 nm assay from Thermo-Fisher, using the smallest possible volume of the sample to minimize interference.
SDS and other detergents will interfere with Lowry and most protein assays, including e.g. Bradford. Either get rid of the SDS or try, for example, the BCA protein assay. It is supposed to be insensitive to SDS.
The Lowry assay is fairly compatible with SDS, but not with so much mercaptoethanol (2% is about 250 mM) or other reducing agents.
Here is a compatibility table for some protein assays:
https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0068-Protein-assay-compatibility.pdf
Leave out the mercaptoethanol if you can. If not, your best bet is probably the 660 nm assay from Thermo-Fisher, using the smallest possible volume of the sample to minimize interference.
Thanks Adam and Sven.... I have an article for reference that shows the compatibility of SDS with lowry assay. But is there any possibility of colour development bcoz of contamination of Blank Buffer? More if I skip the mercaptoethanol then what would be its consequences?
The color development from the blank buffer is probably caused mainly by the mercaptoethanol. If you leave it out, the high blank signal should be greatly reduced.
Thanks adam.. I have tried doing without mercaptoethanol but its lowering the colour intensity of my samples as well. Can you please share some refined protocol for buffer based lysis of yeast cells and protein quantification using lowry assay in a spectrophotometer?
To remove interfering substances prior to Lowry assay, you can use the modification by Bensadoun and Weinstein.
Article Bensadoun A & Weinstein D.Assay of proteins in the presence ...
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