Accurate determination of the protein concentration is essential for most biochemical studies. Bradford protein assay is a rapid and sensitive method. Often interfering substances (detergents, buffers, etc) are essential in the protein sample. If the the concentration of the interfering substance is not known or the blank absorbance is to high, precipitation of the protein is a convenient method to remove interfering material.
The protein determination method of Bradford is rapid and sensitive. Coomassie brilliant blue G-250 exists in three forms: cationic (470-nm red), neutral (650-nm green), and anionic (595-nm blue). The binding of the dye to protein causes a shift in the absorption maximum of the dye from 470 to 595 nm, and it is the increase in absorption at 595 which is monitored.
Preparation of protein reagent:
Dissolve 100 mg Coomassie Brilliant Blue G-250 in 50 ml 95% ethanol. Add 100 ml 85% (w/v) phosphoric acid and 50 ml water. Filter the solution through filterpaper and store at -20°C.
Pipet the protein solution, containing 1 to 25 µg protein, in a volume up to 900 µl, into a plastic micro cuvet. Adjust the volume in the cuvet to 0.9 ml with appropriate buffer or water. Add 0.2 ml of the protein reagent to the cuvet and mix. Measure the absorbance at 595 nm after 2 min and before 1 hour, against a reagent blank prepared from 0.9 ml of the appropriate buffer or water and 0.2 ml protein reagent.
Plot the weight of the reference protein (1 to 25 µg bsa) against the corresponding absorbance resulting in a standard curve. Determine the protein concentration of the unknown samples. [Bradford (1976)][Compton (1985)]