Linearization of the Bradford Protein Assay

Summary

The accuracy and sensitivity of protein determination by the rapid and convenient Bradford assay is compromised by intrinsic nonlinearity. We show a simple linearization procedure that greatly increases the accuracy, improves the sensitivity of the assay about 10-fold, and significantly reduces interference by detergents.

Abstract

Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, the ratio of the absorbance measurements at 590 nm and 450 nm is strictly linear with protein concentration. This simple procedure increases the accuracy and improves the sensitivity of the assay about 10-fold, permitting quantification down to 50 ng of bovine serum albumin. Furthermore, the interference commonly introduced by detergents that are used to create the cell lysates is greatly reduced by the new protocol. A linear equation developed on the basis of mass action and Beer's law perfectly fits the experimental data.

Protocol

Linearization of the Bradford Protein Calibration Graph:

The Coomassie brilliant blue protein assay, commonly known as the Bradford assay ¹, is widely used because of its rapid and convenient protocol as well as its relative sensitivity. Unfortunately, there is a large degree of curvature over a broad range of protein concentrations (Fig. 1). Therefore, only a narrow range of relatively high protein concentrations, 2-10 μg/ml BSA, is used for the calibration graph, which then better fits linear regression (Fig. 1, green). However, the nonlinearity requires protein concentration of the unknown samples to fall within the limited rang....

Discussion

The Bradford protein assay is popular due to its ease of performance and relative sensitivity. The linearization over the entire protein concentrations range obtained by the protocol presented here further simplifies the assay, as the unknown samples do not need to fall within the range of the calibration graph.

Importantly, the improved protocol further provides the following advantages over the original Bradford protocol:

1. Increased accuracy.
2. The sensitivity is incre.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bradford reagent	Bio-Rad	500-0006
Bovine Serum Albumin	Amersco	0332
Multiplate absorbance reader	BioTek	Synergy2