The advantages of using this method are:
- It is very simple.
- The sample can be recovered.
While the disadvantages are:
- Interference from other chromophores.
- The specific absorption value for a given protein must be determined.
- 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:
- Prepare a reliable Spectrophotometer.
- The protein solution must be diluted in the buffer to a concentration that is well within the accurate range of the instrument.
- The protein solution to be measured can be diluted in a wide range of buffers.
- 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.
- 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.
Reference: Number 5 on References