Bradford Reagent
1 of 1
Bradford Reagent (5X concentrate)
100 mg
Coomassie Brilliant Blue G-250
47 ml
Methanol (100%)
100 ml
Phosphoric Acid (85%)
QS to 200 ml with H2O

NOTE: Coomassie must be dissolved in the methanol FIRST before the other
ingredients are added.
For protein concentration analysis
 The BioRad protein assay reagent is the same as the Bradford reagent so we make
our own instead of busying it and it works just as well
 We cut all our volumes in half so use 50 μl of protein dilution and 2.5 ml of
Bradford reagent
 I usually choose 5 μl of protein sample diluted in 45 μl of water as a starting point
because my yields are usually pretty good
 We use a 1 mg/ml BSA solution for our standard
 I have set up an Excel spreadsheet to determine the concentration from the
absorbance
Sample set-up:
Sample
Blank
0.2 mg/ml BSA
0.4 mg/ml BSA
0.6 mg/ml BSA
0.8 mg/ml BSA
Sample 1
Sample 2
Sample 3
Protein
0 μl
10 μl
20 μl
30 μl
40 μl
5 μl
5 μl
5 μl
Water
50 μl
40 μl
30 μl
20 μl
10 μl
45 μl
45 μl
45 μl
Bradford reagent
2.5 ml
2.5 ml
2.5 ml
2.5 ml
2.5 ml
2.5 ml
2.5 ml
2.5 ml
For Excel Spreadsheet:
DF is the dilution factor of your lysate (i.e. 5 μL of lysate would be a DF of 10)
Conc (mg/ml) is the concentration of the diluted sample
T Conc (mg/ml) is the concentration of your lysate
μl/20 μg and μl/50 μg is the amount of lysate needed for 20 μg and 50 μg of protein
repectively
The spreadsheet is set up so that all you have to do is enter the Abs values for your
standards and the Abs and DF values for your samples, then it will calculate the rest.
01/02/07