Bicinchoninic Acid Assay (BCA)

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Introduction

The Bicinchoninic Acid Assay (BCA), also referred to as the Smiths' Assay, is a biochemical assay designed by Paul Smith in 1985. (Smith, et al., [7])  This assay uses colorimetric detection and quantification to determine the total concentration of protein in a solution by exhibiting a measurable color change.

Samples of unknown protein and known relative standard are treated with reagents and the absorbance is recorded from a spectrophotometer at 562 nm. Using a known standard and comparing the unknown to it allows one to determine the concentration based on the curve.

Applications of this assay include studying protein-protein interactions, estimating percent recovery of membrane proteins from cell extracts, high-throughput screening of fusion proteins, measuring column fractions after affinity chromatography and measuring protein covalently bound to agarose supports and protein adsorbed to multiwell plates.

Chemical reaction

The first step in the reaction is the chelation of copper with protein in the alkaline environment  created (sodium potassium tartrate) which results in a light blue complex. This reaction, known as the biuret reaction, allows for peptides containing three or more amino acid residues to form a colored chelate complex with the copper ions.

The reaction of BCA with cuprous ion.  Two molecules of BCA bind to each molecule of copper that have been reduced by a peptide-mediated biuret reaction. ( Thermo Fischer, [5] )

The reaction of BCA with cuprous ion. Two molecules of BCA bind to each molecule of copper that have been reduced by a peptide-mediated biuret reaction. (Thermo Fischer, [5])

In the second step of the reaction, two molecules of Bicinchonininc acid react with the reduced cation formed in step one resulting in the purple color change. This reagent is approximately 100 times more sensitive than the biuret reaction alone. 

The complex created is water-soluble and exhibits a strong linear absorbance at 562 nm. The amount of protein present at this time can be quantified and compared to protein solutions with known concentrations. The key to obtaining accurate results with this assay is to make sure that assay and standards receive identical incubation and temperature conditions. 

Materials

Equipment

  • Disposable 1 mL Plastic Cuvettes
  • Microcentrifuge Tubes
  • Calibrated Pipette
  • Pipette Tips
  • Calibrated Spectrophotometer
  • Timer
  • Water Bath

Reagents

Note: pre-prepared commercialized stocks of reagent A and B can be purchased.

Standard Assay

  • Reagent A
    • Sodium bicinchoninate
    • Sodium carbonate monohydrate
    • Sodium tartrate dihydrate
    • Sodium hydroxide
    • Sodium bicarbonate
    • Distilled water
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  • Reagent B
    • Copper (II) sulfate pentahydrate
    • Distilled water
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  • Standard Working Reagent:
    • Reagent A
    • Reagent B
Screen Shot 2018-06-22 at 12.49.57 PM.png

MICROAssay

  • Reagent A:
    • Sodium carbonate monohydrate
    • Sodium hydroxide
    • Sodium tartrate dihydrate
    • Distilled water
Screen Shot 2018-06-22 at 12.12.23 PM.png
  • Reagent B:
    • Sodium bicinchoninate
    • Distilled water
Screen Shot 2018-06-22 at 12.51.17 PM.png
  • Reagent C:
    • Copper (II) sulfate pentahydrate
    • Distilled Water
Screen Shot 2018-06-22 at 12.52.17 PM.png
  • Standard Working Reagent:
    • Reagent A
    • Reagent B
    • Reagent C
Screen Shot 2018-06-22 at 12.11.52 PM.png

References  

  1. “Bicinchoninic Acid(BCA) Protein Assay.” Geno Technology, G Biosciences, 8 Mar. 2016,  https://www.gbiosciences.com/image/pdfs/protocol/786-570_protocol.pdf.
  2. "Bicinchoninic Acid Protein Assay Kit: Technical Bullet". Sigma Aldrich,  https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/bca1bul.pdf.
  3. “Bio-Protocol - Improve Research Reproducibility.” Bradford Protein Assay -BIO-PROTOCOL, bio-protocol.org/bio101/e44.
  4. Boyer, Rodney. Biochemistry Laboratory: Modern Theory and Techniques. 2nd ed., Pearson, 2012.
  5. “Chemistry of Protein Assays | Thermo Fisher Scientific - US.” Thermo Fisher Scientific, Thermo Fisher Scientific, www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/chemistry-protein-assays.html.
  6. “Pierce BCA Protein Assay Kit.” Thermo Fisher Scientific, Thermo Fisher Scientific, www.thermofisher.com/order/catalog/product/23225?gclid=Cj0KCQjwvLLZBRDrARIsADU6ojCD_LJOswocze-yyY40iNXY1XE_M7gsrbc2W-DvEShra1dHq3zhQ7caAj4qEALw_wcB&s_kwcid=AL%213652%213%21265618211699%21b%21%21g%21%21&ef_id=Wy0z7wAAABlGx4rG%3A20180622173956%3As.
  7. P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H. Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goeke, B.J. Olson, D.C. Klenk, Measurement of protein using bicinchoninic acid, Anal. Biochem. 150 (1985) 76–85. 
  8. Walker J.M. (2002) The Bicinchoninic Acid (BCA) Assay for Protein Quantitation. In: Walker J.M. (eds) The Protein Protocols Handbook. Humana Press. https://doi.org/10.1385/1-59259-169-8:11