How to Calculate Protein Concentration Using The Bradford Assay?
The Protein Man Says:
Before attempting to study enzyme kinetics, determine the protein content of cell fragments, or determine the binding constant of a solution, it’s necessary to start by measuring the protein concentration of the solution in question. Once we know how much protein a solution contains, we can better compare the results of one experiment with another. We can also better study the effects of an outside influence on one protein versus another. To measure and calculate protein concentration, there are three standard methods at our disposal: 1.) Absorbance at 280 nm, 2.) the BCA assay, and 3.) the Bradford Assay.
The Bradford Assay
The choice of assay should be influenced by the nature of the protein in question. The Bradford Assay isn’t a very effective measurement method for acidic or basic proteins, and it doesn’t work very well for histidine, lysine, and phenylalanine residues. But for arginine or aromatic residues, the Bradford assay is usually the assay of choice. This method can be completed quickly, and the reagent (Coomassie Brilliant blue) isn’t usually influence by the presence of mercaptoethanol or other reducing agents that may appear in a buffer, unlike the BCA assay.
The Bradford Assay is colorimetric, so when the Coomassie Brilliant blue binds with the protein residues in the solution, its maximum absorption shifts from 465 to 595 nm. This is a visible color change, so the assay requires a visible light spectrometer with a maximum transmission of at least 595nm.
To create a basic Bradford assay reagent, dissolve 100 mg of Coomassie Brilliant Blue in 50 ml of ethanol, then add 100 ml of phosphoric acid solution and dilute to 1 liter. Meanwhile, prepare protein standards containing 5 to 100 micrograms of albumin or gamma globulin (gamma globulin is preferred). Add 5 ml of dye reagent to each standard and measure absorbance at 595 nm. Once you obtain these measurements, you can use this absorbance to create a standard curve that can be measured against absorption in the unknown solution.
In today's market there are numerous enhanced, ready-to-use Bradford Assays and protein standards available. Although the Bradford assay is more resistant to reducing agents it can still be affected by detergents and other interfering agents. The Protein Man recommends G-Biosciences' CB-X Protein Assay that is an enhanced Bradford assay with a unique clean up step.
For details on the actual calculations and the types of standard curves to use, please see The Protein Man blog on Bradford Protein Assay: Calculation of An Unknown Standard
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