Experiment 4

Theory

Measurement of protein concentration in solution has since long been of essential

importance in biological laboratories. The most commonly used ways to determine protein
concentrations are the Bradford, Lowry and BCA methods. These methods, however, have
definite limitations speaking of sensitivity, dynamic range and in the case of the Bradford and
Lowry assay, compatibility with reducing agents. Even though a majority of biological and
biochemical laboratories has at least at some point applied one of these methods, the detection
and determination of some protein concentrations might not be satisfactory, if possible at all.

The Bradford Reagent can be used to determine the concentration of proteins in solution.
The procedure is based on the formation of a complex between the dye, Brilliant Blue G, and
proteins in solution. The Bradford Reagent is compatible with reducing agents, which are often
used to stabilize proteins in solution. Other protein assay procedures (Lowry and BCA) are not
compatible with reducing agents. The Bradford Reagent should be used in place of these protein
assays if reducing agents are present. However, the Bradford reagent is only compatible with low
concentrations of detergents if the protein sample to be assayed has detergents present in the
buffer, it is suggested to use the BCA protein determination procedure.

The Bradford assay, a colorimetric protein assay, is based on an absorbance shift in the
dye Coomassie when the previously red form commassie reagent changed and stabilized into
coomassie blue by the binding of protein. Two types of bond interaction take place here, the red
form of coomassie dye first donates its free proton to the ionizable groups on protein, which
causes a disrupture of protein's native state, and consequently exposes its hydrophobic pockets.
The exposed hydrophobic pockets on the protein chain will bind non-covalently to the non-polar
region of the dye via van der Waals force, this will position the positive amine groups to
proximity with the negative charge of dye, and the bond is further strengthened by the ionic
interaction between the two. Binding of the protein stabilized the blue form of coomassie dye,
and the amount of the complex present in solution is a measure for the protein concentration by
use of absorbance reading.
 In preparing standard curve, a known protein with different concentrations achieved via a
series of dilution is plotted against their absorbance value. Hence, by measuring the absorbance
of unknown sample, the protein concentration can be predicted.