Colorimetric protein assay techniques

There has been an increase in the number of colorimetric assay techniques f or the determination of protein concentration over the past 20 years. This has resulted in a perceived increase in sensitivity and accuracy with the a dvent of new techniques, The present review considers these advances with e mphasis on the potential use of such technologies in the assay of biopharma ceuticals, The techniques reviewed include Coomassie blue G-250 dye binding (the Bradford assay), the Lowry assay, the bicinchoninic acid assay and the biuret assay. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in the literature , accuracy and reproducibility/coefficient of variation/laboratory-to-labor atory variation. A comparison of the use of several assays with the same sa mple population is presented. It is suggested that the most critical issue in the use of a chromogenic protein assay for the characterization of a bio pharmaceutical is the selection of a standard for the calibration of the as say; it is crucial that the standard be representative of the sample. If it is not possible to match the standard with the sample from the perspective of protein composition, then it is preferable to use an assay that is not sensitive to the composition of the protein such as a micro-Kjeldahl techni que, quantitative amino acid analysis or the biuret assay. In a complex mix ture it might be inappropriate to focus on a general method of protein dete rmination and much more informative to use specific methods relating to the protein(s) of particular interest, using either specific assays or antibod y-based methods, The key point is that whatever method is adopted as the 'g old standard' for a given protein, this method needs to be used routinely f or calibration.