BCA Assay

The BCA Assay is a detergent-compatible, biochemical assay that quantifies protein concentration by measuring the color change of bicinchoninic acid from blue to purple.

The bicinchoninic acid (BCA) protein assay quantifies protein concentration in solution (20 µg / mL to 2000 µg / mL). Like the Lowry protein assay and Bradford protein assay, it is a colorimetric method; it observes the color change of BCA from blue to purple. This shift is directly proportional to the amount of protein and can be measured using absorbance at 562 nm.

Principle and mechanism

Originally invented by Paul Smith at the Pierce Chemical Company, the BCA assay involves a two-step detection process. The first is a Biuret reaction, in which Cu²⁺ ions are reduced to Cu⁺ ions, in an alkaline environment, through the formation of chelate complexes with amino acid residues (cysteine, tyrosine and tryptophan). In the second step, BCA reacts with the cuprous ions produced in step one, forming a water soluble complex with a strong absorbance at 562 nm. This absorbance can be quantified using a microplate reader or other absorbance spectrophotometer, and it is directly proportional to the protein concentration in solution.

Bradford Assay vs BCA

While similar to the Bradford assay, the BCA assay has several key differences. Firstly, it has a broader detection range than the Bradford assay. Second, compared to the Bradford assay, the BCA assay is less sensitive to the specific protein composition of the sample being tested. This is because the peptide backbone, in addition to specific residues, contributes to the final absorbance reading.

A list of additional differences is summarized in the table below:

	Bradford Assay	BCA Assay
Assay Range (µg / mL)	10 to 50 (10 to 500 if using ratiometric method)	20 to 2000
Principle	Coomassie Brilliant Blue-protein complex formation	Copper ion reduction and BCA-Cu+ complex formation
Absorbance (nm)	595	562
Detergent compatibility	Low	High
Protein residues influenced by	arginine, lysine, histidine	cysteine, tyrosine, tryptophan

Micro BCA vs BCA

While both assays use BCA (bicinchoninic acid) to measure protein concentration of dilute protein samples, the Micro BCA assay is designed for testing proteins in lower sample concentrations of 0.5-20 µg / mL, while the standard BCA assay works in the range of 20 - 2000 µg / mL.