Bradford assay protocol

Principle of the Bradford assay

This protein assay is an accurate method for determining a protein concentration in a solution, involving the binding of Coomassie Brilliant Blue G-250 dye to the target protein. This dye can be found in three different forms and colours: cationic (red), neutral (green) and anionic (blue).
When conditions are acidic, it can be found in its double-protonated red cationic form with Amax 470nm, changing to its unprotonated blue state with Amax 595nm when bound to a protein. When in this state, it can be easily detected using a spectrophotometer or microplate reader at 595nm.

Reagents:

Protein:
  • Sample fractions
  • PBS
  • 2 mg/mL of BSA
Bradford assay
  • Bradford reagent (diluted), containing methanol, phosphoric acid and the Coomassie Brilliant Blue G-250 dye.

Materials and equipment

  • Plate reader
  • 96-well plates
  • Centrifuge
  • Tubes

Methods

  • Prepare 7x200uL dilutions from the BSA standard using the PBS buffer.
  • Add 10uL of each dilutions to the 96-well plate with dilutions suitable for the experiment at hand.
  • Use 3 wells with 10uL of PBS as blanks.
  • Introduce 200 uL of the dye reagent to the wells and leave on the underpad for 5 minutes.
  • Measure the absorbance of the plate to obtain data for the standard curve. The intensity of the colour in the well is proportional to the amount of protein present.

Note: Do not include the 200uL of Bradford reagent added to each well in the standard curve calculations. Only the BSA and protein dilutions are needed for the curve.

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