APPENDIX 3B Spectrophotometric Determination of Protein Concentration

  1. Michael H. Simonian

Published Online: 1 AUG 2002

DOI: 10.1002/0471143030.cba03bs15

Current Protocols in Cell Biology

Current Protocols in Cell Biology

How to Cite

Simonian, M. H. 2002. Spectrophotometric Determination of Protein Concentration. Current Protocols in Cell Biology. 15:3B:A.3B.1–A.3B.7.

Author Information

  1. Beckman Coulter Inc., Fullerton, California

Publication History

  1. Published Online: 1 AUG 2002
  2. Published Print: JUL 2002


This unit describes spectrophotometric and colorimetric methods for measuring the concentration of a sample protein in solution. Absorbance measured at 280 nm (A280) is used to calculate protein concentration by comparison with a standard curve or published absorptivity values for that protein (a280). Alternatively, absorbance measured at 205 nm (A205) is used to calculate the protein concentration. The A280 and A205 methods can be used to quantify total protein in crude lysates and purified or partially purified protein. A spectrofluorometer or a filter fluorometer can be used to measure the intrinsic fluorescence emission of a sample solution; this value is compared with the emissions from standard solutions to determine the sample concentration. The fluorescence emission method is used to quantify purified protein. This simple method is useful for dilute protein samples and can be completed in a short amount of time. There are two colorimetric methods: the Bradford colorimetric method, based upon binding of the dye Coomassie brilliant blue to the protein of interest, and the Lowry method, which measures colorimetric reaction of tyrosyl residues in the protein sample.