فروشگاه اینترنتی کالا زیست

BRADFORD PROTEIN ASSAY

 History & Theory

 Four spectroscopic methods are routinely used to determine the concentration of protein in a solution. These include measurement of the protein's intrinsic UV absorbance and three methods which generate a protein-dependent color change; the Lowry assay, the Smith copper/bicinchoninic assay and the Bradford dye assay.

The first, UV absorbance, requires that a pure protein with known extinction coefficient be used, in a solution free of interfering (UV absorbing) substances. The Lowry and copper/bicinchoninic assays are based on reduction of Cu²⁺ to Cu¹⁺ by amides.  Although this makes them potentially quite accurate, they require the preparation of several reagent solutions, which must be carefully measured and mixed during the assay.  This is followed by lengthy, precisely timed incubations at closely controlled, elevated temperatures, and then immediate absorbance measurements of the unstable solutions.  Both assays may be affected by other substances frequently present in biochemical solutions, including detergents, lipids, buffers and reducing agents. This requires that the assays also include a series of standard solutions, each with a different, known concentration of protein, but otherwise having the same composition as the sample solutions.

The Bradford assay is faster, involves fewer mixing steps, does not require heating, and gives a more stable colorimetric response than the assays described above.  Like the other assays, however, its response is prone to influence from non protein sources, particularly detergents, and becomes progressively more nonlinear at the high end of its useful protein concentration range. The response is also protein dependent, and varies with the composition of the protein. These limitations make protein standard solutions necessary.