I have prepared Bradford reagent as follows:
- Dissolve 50mg of Coomassie Blue G250 in 50ml of methanol.
- Add 100ml of 85% H3PO4 to the solution from step 1.
- Add the solution from step 2 into 500ml of H2O and mix.
- Filter to remove and precipitates.
- Add an additional 350ml of H2O.
- Store at 4oC.
HOWEVER, this prepared reagent cannot distinguish between low protein concentrations (0.5 and 0.1 mg/mL) where both concentrations give the same absorbance values.
HOW can I enhance the detection limit of the prepared BRADFORD to be comparable to the commercial one purchased from Bio-Rad?