Determining the identity and purity of recombinant proteins by UV absorption spectroscopy.
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.
Mach
H
H
Merck Research Laboratories, West Point, Pennsylvania, USA.
Middaugh
C R
CR
Denslow
N
N
D002851:Chromatography, High Pressure Liquid; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
D011994:Recombinant Proteins