Quantitation of protein.
The measurement of protein concentration in an aqueous sample is an important assay in biochemistry research and development labs for applications ranging from enzymatic studies to providing data for biopharmaceutical lot release. Spectrophotometric protein quantitation assays are methods that use UV and visible spectroscopy to rapidly determine the concentration of protein, relative to a standard, or using an assigned extinction coefficient. Methods are described to provide information on how to analyze protein concentration using UV protein spectroscopy measurements, traditional dye-based absorbance measurements; BCA, Lowry, and Bradford assays and the fluorescent dye-based assays; amine derivatization and detergent partition assays. The observation that no single assay dominates the market is due to specific limitations of certain methods that investigators need to consider before selecting the most appropriate assay for their sample. Many of the dye-based assays have unique chemical mechanisms that are prone to interference from chemicals prevalent in many biological buffer preparations. A discussion of which assays are prone to interference and the selection of alternative methods is included.
Noble
James E
JE
National Physical Laboratory, Teddington, Middlesex, United Kingdom.
Bailey
Marc J A
MJ
D000465:Algorithms; D000588:Amines / Q000378:metabolism; D000818:Animals; D001671:Biochemistry / Q000295:instrumentation / Q000379:methods*; D003124:Colorimetry / Q000295:instrumentation / Q000379:methods; D006801:Humans; D007202:Indicators and Reagents / Q000138:chemical synthesis / Q000737:chemistry; D008163:Luminescent Measurements / Q000295:instrumentation / Q000379:methods; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D011804:Quinolines / Q000737:chemistry / Q000494:pharmacology; D012394:Rosaniline Dyes / Q000737:chemistry / Q000494:pharmacology; D056928:X-Ray Absorption Spectroscopy / Q000295:instrumentation / Q000379:methods
D000588:Amines; D007202:Indicators and Reagents; D011506:Proteins; D011804:Quinolines; D012394:Rosaniline Dyes; C024795:bicinchoninate reagent; C048139:Coomassie blue