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Bradford Method: Colorimetric Protein Assay


Bradford method is a common colorimetric method to determine protein concentration in a sample solution. The Bradford method of protein determination is based on the binding of a dye, Coomasie Blue G, to the protein. This binding shifts the absorption maximum of the dye from red to blue. The absorbance of the solution is measured at 595 nm and is proportional to protein concentration when compared to a standard curve.


Materials


Coomasie Blue G

Ethanol

Phosphoric acid, 85% – Caution! Phosphoric acid can cause burns!

Test Tubes

Bovine Serum Albumin

Spectrophotometer

Micropipettes and tips

Preparation of the 5x Bradford Reagent


Dissolve 100 mg of Coomasie Blue g in 50 ml of ethanol and 100 ml of phosphoric acid (85%). The mixture is stirred for approximately 10 minutes. The solution is diluted to 200 ml with distilled water and filtered.


Method-Protein Determination by the Bradford Method


  1. A 10 ml aliquot of the Bradford concentrate solution is diluted 1:5 (1x) with water to prepare the working solution.
  2. In separate tubes, prepare Bovine Serum Albumin (BSA) dilutions of 1.2, 1.0, 0.8, 0.6, 0.3, and 0.1 mg/ml from the 10 mg/ml stock.
  3. Label six test tubes 1-6, a seventh tube as “blank” and tubes 8-10 as “unknown”.
  4. To tubes 1-6, add 100 microliters of the 1.2, 1.0, 0.8, 0.6, 0.3, and 0.1 mg/ml BSA solutions, respectively, and 100 microliters of distilled water is added to the blank tube. Analyze in triplicate 100 microliters of the unknown sample of BSA in tubes 8-10.
  5. Add five (5) ml of diluted (1x) Bradford reagent to each of the 10 tubes. Stir and let incubate for 20 minutes.
  6. The Spectrophotometer is set at 595 nm, and the instrument is zeroed against the water blank. The absorbance of the other tubes is then measured at 595 nm. The absorbance of each tube (y-axis) is plottes against the protein mass in micrograms (x-axis) and a straight line is drawn through the points.
  7. The amount of protein in the unknown tubes is determined by its absorbance and comparison to the standard protein curve. The concentration of the solution is calculated by the following equation: Concentration of unknown = mg of Protein determined by assay / 0.1 ml

The Bradford method doesn’t measure the presence of peptide bonds but detects specific amino acids, such as arginine, which is believed to be responsible for the binding of the dye to the protein.