Bradford assay

One way to determine the concentration of your purified or overexpressed protein sample.

1. Remove Bradford reagent from 4 °C and set at room temperature.

2. Prepare 50 μl BSA Standard at 1 mg/ml and at 0.1 mg/ml.

3. Prepare 40 μl 20x dilution of protein sample, or cell extract (this can vary depending on your rough estimation of concentration).

4. Add 800 μl water to 7 cuvettes.

5. Prepare standard cuvettes for

   • 0 mg/ml,
   • 1 mg/ml (10 μl 0.1 mg/ml BSA),
   • 2 mg/ml (20 μl 0.1 mg/ml BSA),
   • 4 mg/ml (4 μl 1 mg/ml BSA),
   • 6 mg/ml (6 μl 1 mg/ml BSA).

6. Prepare experimental cuvettes for 2 μl of sample and 4 μl of sample.

7. Add 200 μl of Bradford reagent to each cuvette and mix well by pipetting. Incubate at room temperature for at least 10 min.

8. Produce standard curve at OD 595nm using cuvettes from step 6.5. Reject standard curve if r2 < 0.95. 9. Determine extract concentration at OD 595nm using cuvettes from step 6.6.