Protein Assay using UV-Spectrophotometer at 280 nm

It is possible to estimate protein concentration in a solution by using simple spectrometer. Absorption of radiation in the near UV (280 nm) by proteins depends on the Tyrosine and Tryptophan content (also to a very small extent on the amount of Phenylalanine and disulfide bond).

The advantages of using this method are:

1. It is very simple.
2. The sample can be recovered.

While the disadvantages are:

1. Interference from other chromophores.
2. The specific absorption value for a given protein must be determined.
3. The extinction of nucleic acid in the 280-nm region may be as much as 10 times that of protein at their same wavelength, and hence, a few percent of nucleic acid can greatly influence the absorption.

Here is the step by step method:

1. Prepare a reliable Spectrophotometer.
2. The protein solution must be diluted in the buffer to a concentration that is well within the accurate range of the instrument.
3. The protein solution to be measured can be diluted in a wide range of buffers.
4. Measure the absorbance of the protein solution at 280 nm, using quartz cuvets or cuvets that are known to be transparent to this wavelength, filled with a volume of solution sufficient to cover the aperture through which the light beam passes.
5. The actual value of UV absorbance for a given protein must be determined by some absolute method, e.g., calculated from the amino acid composition, which can be determined by amino acid analysis. The UV absorbance for a protein is then calculated according to the following formula:
   A280 (1 mg/mL) = (5690Nw + 1280Ny + 120Nc)/M

   where Nw, Ny, and Nc are the numbers of Trp, Tyr, and Cys residues in the polypeptide of mass M and 5690, 1280 and 120 are the respective extinction coefficients for these residues.

Regards