how to enhance the detection limit of home-made Bradford reagent? - (Jan/16/2020 )

Dear All

I have prepared Bradford reagent as follows:

1. Dissolve 50mg of Coomassie Blue G250 in 50ml of methanol.
2. Add 100ml of 85% H₃PO₄ to the solution from step 1.
3. Add the solution from step 2 into 500ml of H₂O and mix.
4. Filter to remove and precipitates.
5. Add an additional 350ml of H₂O.
6. Store at 4^oC.

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?

yobou on Thu Jan 16 10:39:46 2020 said:

Dear All

I have prepared Bradford reagent as follows:

 

1. Dissolve 50mg of Coomassie Blue G250 in 50ml of methanol.
2. Add 100ml of 85% H₃PO₄ to the solution from step 1.
3. Add the solution from step 2 into 500ml of H₂O and mix.
4. Filter to remove and precipitates.
5. Add an additional 350ml of H₂O.
6. Store at 4^oC.

 

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?

Since I am using lysis buffer that contains detegents like SDS or NP40, igpal. I need to know if there is a special modification to Bradford reagent.

Thanks a lot

There are kits which are made to analyze proteins with Bradford reagent in the presence of surfactants

if you look up their info sheets, you may find the references which describe how they deal with the surfactants