ABSTRACT
The determination of protein concentration is a somewhat overlooked procedure that is critical for the determination of the specific biological/therapeutic activity of most biopharmaceuticals, the “standardization” or normalization of samples for proteomic analysis and the comparison of cell homogenates. As such, it is unfortunate that most investigators do not recognize the limitations of the various procedures. The reader is recommended to some recent reviews of protein assay methods.1–3 The purpose of this short section is to describe some commonly used techniques for the determination of protein concentration. Care must be taken with the use of these techniques several of the more frequently used techniques depend on protein quality as well as quantity. Thus the technique which is facile might not be accurate. It is noted that accuracy is an attribute in assay validation while facile is not. There are two issues which are common to any of the below assays. The first is the standard and the second is the solvent. The standard should be representative of the sample; albumin might not be the best choice. The concentration of the standard protein cannot be verified by preparation but must be verified by analysis. In other words, accurate dispensing of the standard protein and subsequent dissolution to a given volume does not ensure an accurate standard. The final concentration of a standard solution must be verified by analysis. For well-characterized proteins it is possible to employ ultraviolet spectroscopy using the known extinction coefficient for the standard protein. Thus the A280 of a 1 mg/mL of bovine serum albumin is 0.66 in a cuvette of 1 cm pathlength [126]. It is important to correct for any light scattering due to aggregated material, dust, etc., by recording the baseline over the range 400 to 310 nm where the protein does not absorb. While this procedure may seem somewhat tedious, it is necessary. It is possible to prepare a standard solution which can be used for a substantial period of time. The standard solution is best stored frozen in small aliquots, each of which is used once to calibrate the assay. The precise storage conditions used would require validation.
