IUG, spring 2015
TMZ

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The Bradford assay involves the binding of Coomassie
Brilliant Blue G-250 dye to proteins (Bradford 1976).
The dye exists in three forms: cationic (red), neutral (green), and anionic (blue) (Compton and Jones 1985).
 under acidic conditions, the dye is predominantly in the doubly protonated red cationic form (Amax = 465 nm).
However, when the dye binds to protein, it is converted to a stable unprotonated blue form (Amax = 595 nm).
 It is this blue protein-dye form that is detected at 595 nm in the assay using a spectrophotometer or microplate reader.

• CBBG primarily responds to arginine residues
(eight times as much as the other listed residues)
• CBBG binds to these residues in the anionic form
Absorbance maximum at 595 nm (blue)
• The free dye in solution is in the cationic form,
Absorbance maximum at 465 nm (red).
• Bradford, MM. A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254. 1976.
• Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990).

The spectrum from unbound (red line) and protein bound
(green line) Coomassie ® Brilliant Blue. After binding the absorbance maximum of the dye shifts from 465 nm to 595 nm.

 Bradford reagent:
Dissolve 100 mg Coomassie Brilliant Blue G-250 in 50 ml 95% ethanol, add 100 ml 85% (w/v) phosphoric acid. Dilute to 1 liter when the dye has completely dissolved, and filter through Whatman #1 paper just before use.
 The Bradford reagent should be a light brown in color.
Filtration may have to be repeated to rid the reagent of blue components.

Test Sample
Blank
BSA Standard - 5 µg/ml
BSA Standard - 10 µg/ml
BSA Standard - 15 µg/ml
BSA Standard - 20 µg/ml
BSA Standard - 25 µg/ml
Protein Sample
Sample Volume,
µl
40
50
50
0
10
20
30
Vol. Water,
µl
800
790
780
770
760
750
750
Vol. Bradford
Reagent,
µl
200
200
200
200
200
200
200

1. Prepare a 4-fold dilution of a 2 mg/ml BSA sample by adding 50 µl of 2 mg/ml BSA to 150 µl of dI water to make
200 µl of 0.5 mg/ml BSA.
2. Generate test samples for blank, BSA standards and the protein sample to be tested according to table 1 in disposable cuvettes.
3. Measure the absorbance of each sample at 595 nm using a
UV-visible spectrophotometer.
4. Plot the absorbance of each BSA standard as a function of its theoretical concentration. The plot should be linear.
5. Determine the best fit of the data to a straight line.
Determine the unknown protein’s concentration.