Tuesday, March 31, 2009

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.
    • It is best to measure absorbance in the range 0.05-1.0. At around 0.3 absorbance, the accuracy is greatest.
    • Bovine serum albumin is frequently used as a protein standard; 1 mg/mL has an A-280 of 0.66.
  3. The protein solution to be measured can be diluted in a wide range of buffers.
    • If the solution is turbid, the apparent A-280 will be increased by light scattering, so it will need filtration (you can use 0.2 micrometers Millipore filter) or clarification by centrifugation. For turbid solutions, a convenient approximate correction can be applied by subtracting the A-310 (proteins do not normally absorb at this wavelength unless they contain particular chromophores) from the A-280.
    • At low concentrations, protein can be lost from solution by adsorption on the cuvet; the high ionic strength helps to prevent this. Inclusion of a nonionic detergent (0.01% Brij 35) in the buffer may also help to prevent these losses.
  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.
    • The presence of nonprotein chromophores (e.g., heme, pyridoxal) can increase A-280. If nucleic acids are present (which absorb strongly at 260 nm), the following formula can be applied.

      Protein (mg/mL) = 1.55 A280 -0.76 A260

      This gives an accurate estimate of the protein content by removing the contribution to absorbance by nucleotides at 280 nm, by measuring the A-260 which is largely owing to the latter. Other formulae (using similar principles of absorbance differences) employed to determine protein in the possible presence of nucleic acids is the following:

      Protein (mg/mL) = (A-235 - A-280)/2.51
      Protein (mg/mL) = 0.183 A-230 - 0.075.8 A-260


Reference: Number 5 on References