Pierce™ BCA Protein Assay Kit

The Thermo Scientific Pierce BCA Protein Assay Kit is a two-component, high-precision, detergent-compatible assay reagent set to measure (A562nm) total protein concentration compared to a protein standard.

Features of the BCA Protein Assay Kit:

• Colorimetric—estimate visually or measure with a standard spectrophotometer or plate reader (562nm)
• Excellent uniformity—exhibits less protein-to-protein variation than dye-binding methods
• Compatible—unaffected by typical concentrations of most ionic and nonionic detergents
• Moderately fast—much easier and four times faster than the classical Lowry method
• High linearity—linear working range for BSA equals 20 to 2000 µg/mL
• Sensitive—detect down to 5 µg/mL with the enhanced protocol

Used in more labs than any other detergent-compatible protein assay, Pierce BCA Reagents provide accurate determination of protein concentration with most sample types encountered in protein research. The Pierce BCA Assay can be used to assess yields in whole cell lysates and affinity-column fractions, as well as to monitor protein contamination in industrial applications. Compared to most dye-binding methods, the BCA Assay is affected much less by protein compositional differences, providing greater protein-to-protein uniformity.

Applications:
• Studying protein:protein interactions
• Measuring column fractions after affinity chromatography
• Estimating percent recovery of membrane proteins from cell extracts
• High-throughput screening of fusion proteins

How the BCA Protein Assay Detects Protein:
The BCA Protein Assay combines the well-known reduction of Cu²⁺ to Cu¹⁺ by protein in an alkaline medium with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu¹⁺) by bicinchoninic acid. The first step is the chelation of copper with protein in an alkaline environment to form a light blue complex. In this reaction, known as the biuret reaction, peptides containing three or more amino acid residues form a colored chelate complex with cupric ions in an alkaline environment containing sodium potassium tartrate.

In the second step of the color development reaction, bicinchoninic acid (BCA) reacts with the reduced (cuprous) cation that was formed in step one. The intense purple-colored reaction product results from the chelation of two molecules of BCA with one cuprous ion. The BCA/copper complex is water-soluble and exhibits a strong linear absorbance at 562 nm with increasing protein concentrations. The BCA reagent is approximately 100 times more sensitive (lower limit of detection) than the pale blue color of the first reaction.

The reaction that leads to BCA color formation is strongly influenced by four amino acid residues (cysteine or cystine, tyrosine, and tryptophan) in the amino acid sequence of the protein. However, unlike the Coomassie dye-binding methods, the universal peptide backbone also contributes to color formation, helping to minimize variability caused by protein compositional differences.