Pierce™ Rapid Gold BCA Protein Assay Kit

The Pierce Rapid Gold BCA Protein Assay Kit is a fast, two-component, high-precision, detergent-compatible assay optimized to determine total protein concentration. It uses the same copper-chelating technology as the well-known traditional Pierce BCA Protein Assay and provides comparable accuracy but with a 5-min, room temperature incubation and measured at 480 nm. This improvement eliminates the need to wait or expose the samples to elevated temperatures for a fast time to results.

Features of the Rapid Gold BCA Protein Assay Kit include:

• Absorbance—colorimetric assay, with optimal absorbance at 480 nm
• Uniformity—exhibits similar protein-to-protein variation as traditional BCA Protein Assay and less protein-to-protein variation than dye-binding protein assay methods (Bradford)
• Compatibility—unaffected by typical concentrations of most ionic and non-ionic detergents
• Assay time—5-min, room temperature reaction
• Assay range—linear working range for BSA of 20 to 2000 µg/mL

The traditional BCA Protein Assay is a widely used and accepted protein assay, trusted for its highly accurate protein concentration determination and compatibility with most sample types encountered in protein research. However, in order to develop the samples in this traditional protocol, one must either heat the reaction at 37°C for 30 minutes or allow room temperature development for two hours. The Rapid Gold BCA Protein Assay maintains the key characteristics of the traditional BCA assay but allows a fast time and room temperature incubation equal to dye-binding methods. The Rapid Gold 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 and similar to the traditional BCA assay, the Rapid Gold BCA assay is affected much less by protein compositional differences, providing low protein-to-protein variation.

How the Rapid Gold BCA Protein Assay detects protein

The Rapid Gold BCA Protein Assay uses the same copper reduction method as the traditional BCA Protein Assay with a unique copper chelator. The 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 the proprietary chelator. The first step is the chelation of copper with protein in an alkaline environment to form a light green 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, the Rapid Gold BCA chelator reacts with the reduced (cuprous) cation that was formed in step one. The intense gold-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 480 nm with increasing protein concentrations. The complex is approximately 100 times more sensitive (lower limit of detection) than the pale green color of the first reaction.

The reaction that leads to the color formation is strongly influenced by four amino acid residues (cysteine, 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.

Compatibility of the Rapid Gold BCA Protein Assay

Certain substances are known to interfere with the Rapid Gold BCA Protein Assay, including those with reducing potential, chelating agents, and strong acids or bases. Because they are known to interfere with protein estimation at even minute concentrations, avoid the following substances as components of the sample buffer:

• Ascorbic acid
• EGTA
• Iron
• Impure sucrose
• Catecholamines
• Impure glycerol
• Lipids
• Tryptophan
• Creatinine
• Hydrogen peroxide
• Melibiose
• Tyrosine
• Cysteine
• Hydrazides
• Phenol Red
• Uric acid