The bicinchoninic acid (BCA) assay is a copper-based colorimetric assay for total protein quantification. The assay relies on reduction of the cupric ions (Cu2 +) to cuprous cation (Cu+) by proteins. Peptides containing three or more amino acid residues form colored chelating complexes with Cu2+ in an alkaline environment, reducing Cu2+ to Cu+. Then BCA reagent as a highly selective colorimetric detection reagent chelate to Cu+ causing a purple color with a strong absorbance at 562 nm. The amount of Cu2 + reduced is proportional to the amount of protein present in the solution. Bicinchoninic-Cu+ complexes are affected by the number of peptide bonds and the presence of cysteine, cystine, tyrosine, and tryptophan side chains. In addition, the development of color in the BCA assay depends on time, temperature, and pH. The assay can be performed at room temperature, but increasing the reaction temperature can significantly reduce the time required for maximum color development and improve sensitivity.
Principle of the Bicinchoninic Acid (BCA) Assay
One of the advantages of the BCA assay is the low protein-to-protein variability. The composition and structure of proteins are diverse, and in some assays, differences in the amino acid sequence, isoelectric point, secondary structure, or side chains result in variations in the colorimetric reaction. This low protein-to-protein variation results in greater accuracy in determining the protein concentration of unknown protein samples. Another advantage of the BCA assay is that it produces a linear response curve that allows accurate determination of unknown protein concentrations and provides a higher dynamic range than other standard assays. In addition, the BCA assay is generally more resistant to the compounds that interfere with the Lowry assay. In particular, BCA assay is compatible with a range of detergents and denaturants such as urea and guanidine chloride.
Amerigo Scientific offers the QPRO kit and μQPRO kit based on the BCA assay for the rapid and highly sensitive colorimetric analysis.
|QPRO - BCA kit Standard for Protein Quantitation||Reagent A 500 mL; Reagent B 15 mL||20-2000 μg/mL protein|
|μQPRO - BCA kit Micro for Protein Quantitation||Reagent A 240 mL; Reagent B 240 mL; Reagent C 12 mL||0.5-20 μg/mL protein|
Reagent A: tartrate in carbonate buffer; Reagent B: 4% BCA in water; Reagent C: 4% CuSO4•5H2O in water
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