Bradford uses Coomasie Blue which is a dye that binds specifically to proteins. It is very accurate and sensitive, compatible with most buffers, sugars, and chaotropic agents but high concentrations of detergent interfere in the assay

1. Determination of protein concentration
by
Bradford Assay
Dr Vijay Hemmadi

2. What is Bradford assay?
• Colorimetric assay
• Spectrophotometric assay
• Quantitative assay
• To measure protein concentration in a solution

3. Aim
To determine the total protein concentration in a given sample
Principle
Proteins +
Coomassie Blue G250 dye
Bradford Reagent- Acidified solution of protonated Coomassie Blue G250 dye --Red in colour-- Cationic

4. pH0 --both the Sulfate groups --
negatively charged –
all three Nitrogens -- positively
charged -- dye +1 net charge
(465nm)
Neutral Green
pH 1-1.5
(620nm)
above pH2
(595nm)
Principle

5. The dye binds more readily to the cationic residues, lysine and arginine
sulfonic acid group binding to positive amines
Principle

7. Principle

9. Bradford reagent:
Bradford reagent is prepared as follows:
1. Weigh 100mg of Coomassie Blue G250 dye and dissolve it in 47ml of
100% Methanol.
2. Mix this solution with 100ml of concentrated (85%) phosphoric acid.
3. Make the final volume of the solution to 200ml by adding distilled water.
4. Filter the reagent through Whatman No. 1 filter paper.
5. Transfer the filtrate in an amber coloured bottle and store at room
temperature.
Preparation

10. 1mg/mL
Measurement of the protein concentration

11. SL. No Std./Sample
(µg/tube)
Amount
(in µL)
Water
(in µL)
Absorbance
(A595 nm)
1 0 0 200
2 10 10 190
3 20 20 180
4 40 40 160
5 60 60 140
6 80 80 120
7 Sample V 200-V
Measurement of the protein concentration

13. Concentration of BSA in µg

14. Bradford assay is suitable for measuring the protein amount ranging from 10 – 100μg
Calculation
Calculate the protein concentration using the following formula:
Protein concentration =
Amount of sample in µg (a)
V (µL)
Therefore protein concentration per ml =
Amount of sample in µg (a) X 1000
V (µL)