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The Assay Kit, based on the method of Bradford, is a simple and accurate procedure for determining concentration of solubilized protein. 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. The Protein Assay is a dye-binding assay in which a differential color change of a dye occurs in response to various concentrations of protein.1 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.2,3,4 The Coomassie blue dye binds to primarily basic and aromatic amino acid residues, especially arginine.5 Spector6 found that the extinction coefficient of a dye-albumin complex solution was constant over a 10-fold concentration range. Thus, Beer’s law may be applied for accurate quantitation of protein by selecting an appropriate ratio of dye volume to sample concentration.

For standard assay

Spectrophotometer set to 595 nm

Cuvettes with 1 cm path length matched to laboratory spectrophotometer.

Vortex mixer

Whatman #1 filter (or equivalent) and funnel for dye reagent preparation

Graduated cylinders, pipets, and containers for reagent preparation and storage

Pipets accurately delivering 100 μl and 5.0 ml

For microplate assay

Microtiter plates

Pipets accurately delivering 200 μl and 800 μl

Spectrophotometer set to 595 nm

Cuvettes

Typical standard curve for the Assay kit, bovine serum albumin O.D.595 corrected for blank :200-900 μg/ml x 0.1 ml = 20-90 μg protein.  Standard procedure : 20-100 mg

Typical standard curve for the Protein Microassay (1-20 μg/ml), bovine serum albumin. O.D.595

corrected for blank. 1.25-10 μg/ml x 0.8 ml = 1-8 μg protein.

Standard Protocol

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

    deionized (DDI) water. 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.

    2. 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.

3. Pipet 100 μl of each standard and sample solution into a clean, dry test tube. Protein solutions are

normally assayed in duplicate or triplicate.

4. Add 5.0 ml of diluted dye reagent to each tube and vortex.

5. 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.

6. Measure absorbance at 595 nm.