Protein Quantitation Assays

Bio-Rad Assay

The Bio-Rad Protein Assay is based on the method of Bradford. It involves the addition of an acidic dye to protein solution, and subsequent measurement at 595 nm with a spectrophotometer or microplate reader. Comparison to a standard curve provides a relative measurement of protein concentration.

Principle

The Bio-Rad Protein Assay is a dye-binding assay in which a differential color change of a dye occurs in response to various concentrations of protein. The absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 dye shifts from 465 nm to 595 nm when binding to protein occurs. The Coomassie blue dye binds to primarily basic and aromatic amino acid residues, especially arginine. Beer's law may be applied for accurate quantitation of protein by selecting an appropriate ratio of dye volume to sample concentration. Interferences may be caused by chemical-protein and/or chemical-dye interactions. Basic buffer conditions and detergents interfere with this assay.

Method

  1. Prepare dye reagent by diluting 1 part Dye Reagent Concentrate with 4 parts distilled, deionized H2O.

  2. Filter through Whatman #1 filter (or equivalent) to remove particulates. This diluted reagent may be used for approximately 2 weeks when kept at room temperature.

  3. Prepare three to five dilutions of a protein standard, which is representative of the protein solution to be tested. The linear range of the assay for BSA is 0.2 to 0.9 mg/ml, whereas with IgG the linear range is 0.2 to 1.5 mg/ml.

  4. Prepare five-fold dilutions of your protein sample whose concentration is to be determined.

  5. Pipet 10 ul of each standard and sample solution into a 1.5 ml eppendorf. Protein solutions are normally assayed in duplicate or triplicate.

  6. Add 1.0 ml of diluted dye reagent to each tube and vortex.

  7. Incubate at room temperature for at least 5 minutes. Absorbance will increase over time. Samples should incubate at room temperature for no more than 1 hour.

  8. Measure absorbance at 595 nm.