I have read the protocol of the protein estimation by Lowry's method from many sources such as theses, published papers, etc. The entire protocol is similar except the nm in which the colour to be read. In some protocol, it is mentioned as 750nm and in some others as 720 or 640 or 620. Due to this discrepancy, I referred the original Lowry (1951) paper, in which it has been mentioned about two nanometers to read absorbance, i.e. 750nm (5 to 25 microgram protein/ml) and 500nm (stronger solution).
As a beginner, I am confused on how to select the nanometer length which is ideal to read the absorbance of the colour solution concerning Lowry's method for protein estimation.
Hi Annadurai, I can understand your confusion.
The discrepancy could be due to other assay procedures that have modified the original Lowry procedure of reacting peptide bonds with Cu ions at high pH. (Arying the pH of the reaction can give a violet to blue product)
Other protein assays use the same principle, but have added some modifications, e.g.Bicinchoninic acid assay (from Pierce), Biuret test and the Folin–Ciocalteu reagent. They absorb at different wavelengths.
You can always verify the detection wavelength - just make spectrum scan and select the maximum wavelength for your measurement.
In case of Lowry method I've found that in the range 650-750 nm none of wavelength is crucial.
This reagent interacts with the cuprous ions and the side chains of tyrosine, tryptophan, and cysteine to produce a blue-green color that can be detected between 650 nm and 750 nm. The protein detection range is 5–100 μg.
In our lab we follow 740nm for ideal absorbance for Lowry method
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