The BCA Assay - Commonly used protein assay methods

References: 

P.K. Smith et al. (1985) Anal. Biochem. 150: 76.

K. J. Wiechelman et al. (1988) Anal. Biochem.  175: 231 (This describes the mechanism of the assay)

Quick Guide

How does it work?

• The first step is a Biuret reaction which reduces Cu⁺² to Cu⁺¹
• In the second step BCA forms a complex with Cu⁺¹ which it purple colored and is detectable at 562 nm

Detection Limitations

• 0.2-50 µg

Advantages

• Less susceptible to interference from common buffer substances
• Very sensitive and rapid if you use elevated temperatures
• Compatible with many detergents
• Working reagent is stable
• Very little variation in response between different proteins
• Broad linear working range

Disadvantages

• The reaction does not go to completion when performed at room temperature or 37^oC.  This can be a problem if you are assaying a large number of proteins
• Dilution is often necessary for concentrated protein samples

General Considerations

• This method is related to the Lowry method.  However, the use of BCA instead of the Folin-Ciocalteu reagent, makes it less susceptible to interference
• Since reduced copper is detected in the procedure, make sure that the distilled water used in the procedure is fed from plastic lines and not copper lines.  In general water from 18 megaohm water polishers is satisfactory

Procedure

Bicinchoninic Acid Solution

Commercially available (e.g. Sigma B 9643).  Contains BCA, sodium carbonate, sodium tartrate and sodium bicarbonate in 0.1 M NaOH (pH 11.25)

Copper Sulfate Solution

4% CuSO₄*5H₂O

• Prepare the required amount of protein determination reagent by adding 1 volume copper sulfate solution to 50 volumes of bicinchoninic acid solution.
• Set up test tubes containing samples and known amounts of bovine serum albumin in the range of 0 to 100 micrograms.  Each tube should contain 0.1 mL total volume.
• Add 2.0 mL of the protein determination reagent to each tube and vortex.
• Incubate the tubes at 37°C for 30 min (Alternatively 2h at room temperature, or 15 minutes at 60^oC).
• Cool the tubes to room temperature and determine the absorbance at 562 nm.  After cooling to room temperature, the blank continues to increase in absorbance at approximately 2.3% per 10 min (if you use 60^oC incubation temperature, the absorbance will not increase appreciably).  Because of the small volume in each tube, cuvettes with an approximate 1 mL capacity should be used for the absorbance measurements.

Discussion

This assay measures the formation of Cu⁺¹ from Cu⁺² by the Biuret complex in alkaline solutions of protein.  It was originally thought that the mechanism of the assay was the same as in the Lowry assay, but it has since been determined that there are two distinct reactions that take place with copper ions unique to the BCA assay.  The first reaction occurs at lower temperatures and is the result of the interaction of copper and BCA with the following residues: cysteine, cystine, tryptophan and tyrosine.  At elevated temperatures it has been shown that the peptide bond itself is responsible for color development.  This is why performing the assay at 37^oC or 60^oC increases the sensitivity and reduces the variation in the response of the assay to protein composition.

The Structure of BCA

The BCA reagent replaces the Folin-Ciocalteu reagent used in the Lowry assay with bicinchoninic acid.  The BCA reagent forms a complex with Cu⁺¹, which has a strong absorbance at 562 nm.  BCA is advantageous in that it does not interact with as many substances as the Folin-Ciocalteu reagent, especially detergents and buffers.  The BCA assay is limited in that it interacts with most reducing agents and copper chelators.  In general, these are not critical components of buffers and can be easily eliminated prior to assay.

Typical Standard Curve for a BCA Assay