Quantifying proteins using the Bradford method

The Bradford method is a quantitative protein assay method, based on the binding of a dye, Coomassie Brilliant Blue, to a protein sample, and comparing this binding to a standard curve generated by the reaction of known amounts of a standard protein, usually BSA.

For this assay, protein samples should be diluted in an appropriate buffer (generally the same buffer in which they are dissolved). The BSA standard curve should be prepared using the same buffer.

Materials required

• BSA standard solution (1 mg/ml)
• Bradford assay dye reagent (available commercially, e.g., Bio-Rad Protein Assay Dye Reagent Concentrate, cat. no. 500-0006)
• Protein dilution buffer

Note: Proteins should be diluted in the buffer in which they are dissolved. Use the same buffer to prepare the standard curve.

Note: Concentration of the BSA standard solution should be measured photometrically. A 1 mg/ml solution of BSA should have an A280 of 0.66.

1. Prepare a standard curve by pipetting together carefully the solution volumes listed in the table “Standard curve samples for Bradford protein assay” corresponding to 0, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg/ml BSA. The end volume of all samples should be 200 µl.
2. Dilute an aliquot of the dye reagent concentrate 1:5 with distilled water. Pipet 20 µl of each BSA dilution into a plastic cuvette, add 1 ml diluted dye reagent, mix well, and incubate for 5 min at room temperature.
   Note: Stored in the dark, diluted dye reagent is stable for 2 weeks. Write the date of preparation on the bottle and cover with aluminum foil.
3. Measure the OD at 595 nm for each sample, and plot the standard curve.
4. Prepare a second standard curve by pipetting together carefully the solution volumes listed in the table Standard curve samples for Bradford protein assay corresponding to 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg/ml BSA. The end volume of all samples should be 200 µl.
5. Dilute an aliquot of the dye reagent concentrate 1:5 with distilled water. Pipet 20 µl of each BSA dilution into a plastic cuvette, add 1 ml diluted dye reagent, mix well, and incubate for 5 min at room temperature.
6. Measure the OD at 595 nm for each sample, and plot the standard curve.
7. Prepare dilutions of the protein sample of interest and test 20 µl aliquots in duplicate as above. It is important that the protein samples to be tested are handled in exactly the same manner as the samples used in generating the standard curves. Calculate the protein concentration of the test samples by comparing the OD595 with the standard curve(s), taking into account dilution factors (see figure Standard curve generated using the Bradford method).

Standard curve samples for Bradford protein assay