Protein concentration is a crucial parameter affecting the efficacy of a therapeutic. Accurate quantification provides key information supporting formulation studies and the determination of enzyme kinetics. Quantitation is critical for a wide variety of downstream applications in the preparation of commercial pharmaceuticals. Several approaches which are commonly applied to determine the protein concentration include UV absorption, the Bradford assay and the bicinchoninic acid (BCA) assay. Among these, UV absorption is widely applied due to its relative high accuracy, low cost and short analysis time.
For UV absorption, the protein concentration is determined based on the measurement of the UV absorbance at a characteristic wavelength using the known protein extinction coefficient (Beer’s Law). The strong UV-light absorption of the protein is due to the presence of aromatic side chains in the amino acids, such as tyrosine, tryptophan and cysteine. The absorbance measurement is usually conducted at 280 nm to reduce interferences from other compounds in the protein solutions. According to Beer’s Law, the molar extinction coefficient (M-1cm-1) is a constant for a given substance dissolved in a given solution and measured at a given wavelength. For proteins, percent solution extinction coefficients (g/100mL)-1cm-1 are used for convenience instead of molar extinction coefficients. The percent solution extinction coefficient of a protein is a key parameter which can be determined by calibration with a commercial standard, estimated based on the chemical properties of the protein or it can be obtained from a database such as that provided by NIST.
Protein Analysis with Jordi Labs
Jordi Labs has extensive experience conducting UV-vis absorption and protein analysis. We offer high-quality services for the determination of the protein extinction coefficient and protein solution concentration. Our highly skilled chemists provide outstanding customer service and have the background needed to assist with simple or complex projects.