Bio-Rad Protein Assay - Manual (Page 3)

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Section 1
Introduction
The Bio-Rad Protein Assay, based on the method of Bradford, is a
simple and accurate procedure for determining concentration of solubi-
lized protein. It involves the addition of an acidic dye to protein solution,
and subsequent measurement at 595 nm with a spectrophotometer or
microplate reader. Comparison to a standard curve provides a relative
measurement of protein concentration.
1.1 Principle
The Bio-Rad Protein Assay is a dye-binding assay in which a differ-
ential color change of a dye occurs in response to various concentrations
of protein.
1 The absorbance maximum for an acidic solution of
Coomassie ® Brilliant Blue G-250 dye shifts from 465 nm to 595 nm when
binding to protein occurs. 2,3,4 The Coomassie blue dye binds to primarily
basic and aromatic amino acid residues, especially arginine. 5 Spector 6
found that the extinction coefficient of a dye-albumin complex solution
was constant over a 10-fold concentration range. Thus, Beer’s law may be
applied for accurate quantitation of protein by selecting an appropriate
ratio of dye volume to sample concentration.
Interferences may be caused by chemical-protein and/or chemical-dye
interactions. Table 1 lists those chemical reagents not directly affecting
the development of dye color. (Note: Basic buffer conditions and deter-
gents interfere with this assay.) Since every protein-chemical reagent
combination has not been assayed, it is possible that some of the listed
reagents produce interference in combination with certain proteins.
However, with respect to proteins such as bovine serum albumin and
gamma globulin, the listed reagents show little or no interference. The
acceptable concentrations of reagents for the Standard Procedure are
shown in Table 1. Equivalent concentrations of reagents for the
Microassay Procedure (see Section 2) are 1/40 of those listed in this table,
due to the difference of sample-to-dye ratios between the Standard and
Microassay Procedures.
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