Why do we read the Biuret assay at 540 nm???

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The reason for choosing 540 nm as the wavelength for the Biuret Assay is fairly straightforward.

The reagent itself is already colored blue. In the presence of protein the color shifts toward violet and becomes darker. If we determine the absorbance spectrum of the reagent and then determine the absorbance spectrum of the protein complex, we find that the greatest DIFFERENCE between the two spectra occurs at 540 nm. Reading at this wavelentg then gives the most sensitive assay, the one that gives the maximum response for unit of protein.

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Mike Dalbey

dalbey@biology.ucsc.edu

9/2/97