BCA Protein Dual Range Colorimetric Detection Kit

Assay Principle: 

The DetectX® BCA Protein Dual Range Colorimetric Detection Kit quantitatively measures total protein levels in serum, plasma, urine, cell lysates, and tissue homogenates. This detection kit has a straightforward procedure and incubation time of 2 hours. Users should consult the complete kit insert for instructions before use. The kit includes a Bovine Serum Albumin Standard to create an accurate standard curve.

Protocol Summary:

• Transfer standards or diluted samples into the included transparent microtiter plate.
• Add BCA Color Solution to each well, ensuring even mixing of reagents.
• Incubate the mixture at 37°C for 2 hours, allowing the color-generating reaction between the BCA Color Reagent and the proteins.
• After incubation, use a plate reader to detect the colorimetric signal at 560nm. Calculate protein concentration based on the intensity data and the standard curve.

Background:

Protein determination is one of the most common operations performed in biochemical research. The principle behind the widely-used bicinchoninic acid (BCA) assay is similar to that of the Lowry assay, relying on the formation of a Cu2+-protein complex under alkaline conditions, followed by the reduction of Cu2+ to Cu1+. This reduction is proportional to the protein concentration. Amino acids like cysteine, cystine, tryptophan, tyrosine, and peptide bonds can reduce Cu2+ to Cu1+. In the presence of BCA, the Cu1+ forms a purple-blue complex in alkaline environments, providing the basis for colorimetric detection.

This method combines the biuret reaction (reduction of Cu2+ to Cu1+ by protein in an alkaline medium) with a unique bicinchoninic acid reagent. It offers a highly sensitive and selective colorimetric detection of the cuprous cation (Cu+1). The chelation of two BCA molecules with one cuprous ion forms a purple-colored reaction product, resulting in a water-soluble complex with strong absorbance at 562 nm. The DetectX® BCA Protein Detection Kit demonstrates nearly linear absorbance with increasing protein concentrations over a wide range (6-1,000 µg/mL), making it suitable for a broad spectrum of research applications where protein quantification is required.