Question

What is the advantage of using max wavelength rather than the wavelength nearby such as 580 nm?

This is for the Bradford protein assay where the maximum of the absorbance spectrum is at 595 nm.

Answer 1

The Bradford protein assay is a spectroscopic analytical method used to measure the concentration of protein in a solution.  It   dependents on the amino acid composition of the measured protein. It is a colorimetric protein assay. The method is based on the proportional binding of the dye Coomassie to proteins.Under acidic conditions the red form of the dye is converted into its bluer form, binding to the protein being assayed. There is a shift in the peak absorbance of the dye when it binds to a protein. Unbound Coomassie Blue absorbs light maximally at a wavelength of 465 nm, while the absorption maximum is at 595 nm when the dye is bound to protein.

The binding of protein to Coomassie Blue G250 may shift the absorbance maximum of the blue ionic form of the dye from 590 nm to 620 nm. It might, therefore, appear more sensible to measure the absorbance at the higher wavelength. However, at the usual pH of the assay, an appreciable proportion of the dye is in the green form (λmax = 650 nm) which interferes with absorbance measurement of the dye–protein complex at 620 nm. Measurement at 595 nm represents the best compromise between maximizing the absorbance due to the dye–protein complex while minimizing that due to the green form of the free dye .