BCAassays

BCA assays, or bicinchoninic acid assays, are colorimetric methods for determining total protein concentration in a sample. They are widely used in biochemistry and molecular biology because they provide a relatively simple, reliable measure of protein content.

Principle and chemistry: Under alkaline conditions, peptide bonds and amino acids reduce Cu2+ to Cu+. The resulting cuprous ions (Cu+) complex with bicinchoninic acid to form a stable purple color whose absorbance is measured at 562 nm. The color intensity is proportional to the amount of protein in the sample over a defined linear range.

Format and procedure: Most commercial kits use two reagents—Reagent A containing copper sulfate and Reagent B containing bicinchoninic acid in a buffered solution. The working reagent is prepared by mixing A and B. Samples and standards are incubated with the working reagent for a period (commonly about 30 minutes) at room temperature or 37°C, then the absorbance is read at 562 nm. The method is adaptable to microplate and cuvette formats.

Standards and range: A standard curve is generated with a protein standard such as bovine serum albumin (BSA) or bovine gamma globulin. The BCA assay typically offers a wide linear range, approximately from tens to thousands of micrograms per milliliter, depending on protocol and instrument.

Advantages and limitations: The BCA assay is relatively tolerant of many detergents and reducing agents compared to some other protein assays, making it versatile for complex samples. However, strong reducing agents (for example, DTT or beta-mercaptoethanol at high concentrations), chelating agents like EDTA, or some pigments can interfere with the measurement. Proper controls and matching sample buffers to standards improve accuracy.

Applications: It is widely used for quantifying protein in cell lysates, tissue extracts, purified proteins, and other biological samples where a broad dynamic range and compatibility with common reagents are advantageous.