UNIT 7.2 Determining the Identity and Purity of Recombinant Proteins by UV Absorption Spectroscopy
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
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.
- Published Online: 1 MAY 2001
- Published Print: SEP 1995
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.