Spectrophotometric analyses of proteins are commonly used. UV-VIS spectroscopy is typically used for the determination of protein concentration. Protein concentration is determined either by a dye-binding assay (e.g., the Bradford or Lowry method) or by determining the absorption of a solution of protein at one or more wavelengths in the near UV region (260-280 nm). Another spectroscopic method used in the early phase characterization of biopharmaceuticals is circular dichroism (CD).The Bradford method, which is more sensitive and less affected by most common detergents or other common biochemicals than the Lowry method, is the most widely used dye-binding method. There are two common Bradford methods: the standard assay Bradford method, with a range of 10 to 100 mg, and the microassay method, which is linear between 1 and 10 mg. A standard curve is constructed with a common protein that is readily available in pure form such as bovine serum albumin or bovine gamma globulin. The standards and the sample are then reacted with a solution of Coomassie Brilliant Blue G250 in an acidic solution, and the absorbance measured at 595 nm. The protein concentration of the sample is then calculated against the constructed curve. This value is an approximation of the protein concentration because different proteins react differently with the Bradford reagent. Further on in development the calibration curve should be determined using the protein of interest.
Direct determination of the absorbance of a protein solution requires no other reagents or standards. Two solutions are prepared, one of the sample and one blank solution of water or containing all the buffer components. After zeroing the spectrophotometer at the wavelengths to be measured using the blank solution, the analyst measures the absorbance of the protein solution. For relatively pure solutions, measuring the absorbance at 280 nm (A280) is usually sufficient. However, for protein solutions containing significant amounts of nucleic acid (as little as a few percent), it is best also to determine the absorbance at 260 nm (A260), to correct for the presence of nucleic acids. The protein concentration is then determined using the following equation:
Protein (mg/mL) = 1.55 (A 280 ) – 0.76(A 260 ).
If the extinction coefficient has not yet been determined, a more absolute concentration is determined using the following equation:
A 280 (mg/mL) = (5690 * # Trp + 1280 *# Tyr + 120* #Cys)/ protein MW,
Where #Trp is the number of tryptophan residues in the protein and similarly, #Tyr is the number of tyrosines, #Cys is the number of cysteine residues, and MW is the molecular weight of the protein.