Summary of Bradford assay

The Bradford method is an important method of protein analysis primarily due to its convenience and
high sensitivity. This method is able to detect most proteins easily while requiring one simple dye
reagent and color development is generally rapid.

The usual reagent contains the ethanol, the dye (Coomassie Brilliant Blue G-250) and a significant
amount of phosphoric acid. Phosphoric acid ensures that the standard assay is run at low pH, generally
below pH 1. The assay is performed by adding a protein sample to a fixed quantity of assay reagent
and recording the absorbance. The formation of a blue dye-protein complex is usually apparent on
visual inspection of the mixture.

The Coomassie Brilliant Blue G-250 dye exists in three ionic

forms: anionic, neutral, and cationic. These different charged
states give rise to the different colors of the dye molecule. In the
red form, all three nitrogen atoms carry a positive charge. The
two sulfonic acid groups have extremely low pKa and are in
negatively charged state. Thus at a pH of around zero, the dye
will be a cation with an overall charge of +1 with an absorption
maximum at a wavelength of 465 nm. At a pH of around 1, the
dye is green with an absorption maximum at 620 nm. The green
color corresponds to a form of the dye with no net overall
charge. While above pH 2 the dye is bright blue with a maximum
at 595 nm. Only the nitrogen atom of Fig: Chemical structures of
the diphenylamine moiety carries a positive charge and the blue Coomassie brilliant blue G-250.
dye molecule is an anion with an overall charge of −1.

Under the acid conditions, the dye reagent is normally brownish in color but on binding to the protein,
the blue form of the dye is produced. The dye forms a strong, noncovalent complex with the protein.
During the formation of this complex, the red form of Coomassie dye first donates its free electron to
the ionizable groups (arginyl and lysyl) residues on the protein, which causes a denaturation of the
protein's native state subsequently exposing its hydrophobic pockets. This results in the non-covalent
binding of its pockets to the non-polar region of the dye via the van der Waals forces. This also causes
the alignment of the positive amine groups in proximity with the negative charge of the dye. This ionic
interaction additionally strengthen the complex formed. Thus binding of the protein stabilizes the
blue form of the Coomassie dye, even under acid conditions when most of the molecules in solution
are in the cationic form. As a result, the increase in absorbance at 595 nm is proportional to the
amount of bound dye, and thus to the amount (concentration) of protein present in the sample.

If there is no protein to bind, then the solution will remain brown. The protein-dye complex is
incubated at room temperature for at least 5 min or for no more than 1 hrs., before measuring the
absorbance.
Limitations of Bradford method

1. The dye bind most readily to arginyl and lysyl residues of proteins. This specificity can lead to
variation in the response of the assay to different proteins, which is the main drawback of the
method.
 2. The dye is unable to bind to free arginine or lysine, or to peptides smaller than 3kDa. Many
peptide hormones and other important bioactive peptides fall into this category. The Bradford
assay is not suitable for quantifying those compounds.

3. The assay is linear over a short range from 0 µg/mL to 2000 µg/mL. So there is often necessity
of dilution of the samples before analysis

4. The Bradford assay is relatively free from interference. However, a few chemicals alter the
response of proteins to the dye eg. detergents and ampholytes.

References
1. Bradford MM. (1976) A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72,248–
254.
2. Chial HJ, Thompson HB, Splittgerber AG. (1993 A spectral study of the charge forms of
Coomassie blue G. Analyt. Biochem. 209,258–266.
3. Chial HJ, Splittgerber AG. (1993) A comparison of the binding of Coomassie brilliant blue to
proteins at low and neutral pH. Analyt. Biochem. 213,362–369.
4. Compton SJ, Jones CG.( 1985) Mechanism of dye response and interference in the Bradford
protein assay. Analyt. Biochem. 151, 369–374.
5. Congdon, R. W., Muth, G. W., and Splittgerber, A. G. (1993) The binding interaction of
Coomassie Blue with proteins. Analyt. Biochem. 213, 407–413.