Unit

UNIT 7.2 Determining the Identity and Purity of Recombinant Proteins by UV Absorption Spectroscopy

  1. Henryk Mach1,
  2. C. Russell Middaugh1,
  3. Nancy Denslow2

Published Online: 1 MAY 2001

DOI: 10.1002/0471140864.ps0702s01

Current Protocols in Protein Science

Current Protocols in Protein Science

How to Cite

Mach, H., Middaugh, C. R. and Denslow, N. 2001. Determining the Identity and Purity of Recombinant Proteins by UV Absorption Spectroscopy. Current Protocols in Protein Science. 1:7.2:7.2.1–7.2.21.

Author Information

  1. 1

    Merck Research Laboratories, West Point, Pennsylvania

  2. 2

    University of Florida, Gainesville, Florida

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: SEP 1995

Abstract

Because each protein (gene product) has a unique amino acid sequence, the particular aromatic amino acid content of each protein results in a unique spectrum in the near-UV (250 to 350 nm) region. The highly specific microenvironment experienced by each aromatic residue in the three-dimensional protein matrix results in fine shifts in a protein's spectrum. This unit provides protocols for the detection and analysis of UV spectra of recombinant proteins and their peptide fragments. The unique UV spectral properties of proteins can in turn be used to assess their purity. This application is inherent in the use of a diode array detector to monitor the effluent from a high-performance liquid chromatography (HPLC) column. A protocol using this technique to assess the purity of recombinant proteins is presented.