Protein Assay Kit From Bio-Rad

As accurate quantification of protein concentration in a sample is an important pre-requisite for SDS-PAGE gels and Western blotting, a simple, easy system to compare protein levels between different cell lysates is needed. The Bio-Rad Protein Assay Kit provides just such a system. The principle is quite simple and is modelled on the standard “Bradford Assay” in that the protein quantification solution contains different forms of dye (Coomassie Blue), which change color upon binding to protein. In an acidic environment, the dye appears reddish-brown, but when in a basic state, as when bound to a basic protein, the dye appears blue. The equilibrium between unbound, red dye and bound, blue dye determines the relative concentration of protein.

Immediately prior to use, the assay solution is diluted 1:5 with distilled H₂O. It is important to ensure the water used is of a high purity, as some chemicals (especially basic buffers, detergents and sodium hydroxide) may cause color change, resulting in a higher-than-actual determination of protein concentration. Additionally, as the buffer(s) used for initial cell lysis and protein isolation may also provide background equilibrium state-changes in the quantification solution, it is important to use either small volumes of lysate in the reaction or ensure that the buffers do not cause color changes. We routinely add 2-5 ul of protein lysate to 200 ul of diluted Bio-Rad protein assay solution in a flat-bottomed (not U-bottom) 96-well plate. The color change is measured by a spectrophotometer at a wavelength of 595 nm.

It is important to prepare accurate reference standards for determining absolute protein concentration. We routinely use molecular-grade BSA (available at a concentration of 10 ug/ul with most restriction enzymes) in concentrations ranging from 0.0625 ug/ul to 2 ug/ul to generate our standard curve, as most of our protein lysates are at a concentration within this range. A key consideration, therefore, is to ensure that the assay is not over-saturated with protein as this will not provide accurate linear quantification. High-concentration protein lysates may need to be diluted before use.

In summary, the Bio-Rad Protein Assay Kit provides a simple and convenient reagent to measure protein concentration. Through the use of appropriately chosen protein extraction buffers, and an accurate standard curve, we have found that protein concentrations obtained using this assay are verified by immunoblotting for a housekeeping protein, such as actin or â-tubulin, in our SDS-PAGE experiments.