There has been a need for accurate estimation of sample protein concentration for many years. This was true during the development of early biochemical techniques, such as enzyme reactions, and later for assay of protein concentration in clinical samples such as blood and urine. By necessity, rigorous methods were developed that employed laboratory spectrophotometers and multiplate readers.
Simple absorbance at 280 nm was useful, although relatively insensitive, and is still used today for some applications. Several quantitative assays are now available that bring a high degree of precision and sensitivity. Assay reagents include dye-protein binding, such as Coomassie/Bradford reagents, or copper chelation, as in Lowry and Bicinchoninic Acid (BCA).
Each method has its advantages depending on the time required, possible interfering substances (buffers, salts, detergents, or oxidizing agents), the concentration range, and protein-protein variation. Currently, each assay requires a daily preparation of a standard curve as well as negative controls, whether the analyst was testing one unknown sample or multiple samples.
As the field of biotechnology continues to develop, protein assays play an ever increasing role in protein drug development, R&D, and clinical screening, as well as process development, quality control, and cleaning validation. The need for rapid information at each step in these areas has created the necessity for speed, which in turn has moved the location that protein assays have been performed closer to the point of use.
For instance, in the research lab, rapid feedback on separation column fractions may influence what and how the next steps may be performed. On the production floor, assessment of key samples during fermentation could affect the production schedule as well as quality control. In this article, we will concentrate on the BCA assay and a new method to kinetically read the assay, bringing protein assays closer to point-of-use.