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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 AssayBCA Assay
Assay Range (µg / mL)10 to 50
(10 to 500 if using ratiometric method)
20 to 2000
PrincipleCoomassie Brilliant Blue-protein complex formationCopper ion reduction and BCA-Cu+ complex formation
Absorbance (nm)595562
Detergent compatibilityLowHigh
Protein residues influenced byarginine, lysine, histidinecysteine, 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.

BCA assay protocol

Quick summary

  1. Prepare BCA working solution (50 µL)
  2. Add BSA standards or test samples (50 µL)
  3. Incubate at room temperature for 20 - 60 minutes
  4. Read absorbance at 562 nm (in the range of 540-590 nm)

Full protocol

Citations

View all 1 citations: Citation Explorer
Increased alcohol dehydrogenase 1 activity promotes longevity
Authors: Ghaddar, Abbas and Mony, Vinod K and Mishra, Swarup and Berhanu, Samuel and Johnson, James C and Enriquez-Hesles, Elisa and Harrison, Emma and Patel, Aaroh and Horak, Mary Kate and Smith, Jeffrey S and others,
Journal: Current Biology (2023)

References

View all 15 references: Citation Explorer
Specialized Cell-Free DNA Blood Collection Tubes Can Be Repurposed for Extracellular Vesicle Isolation: A Pilot Study.
Authors: Heatlie, Jessica and Chang, Vanessa and Fitzgerald, Sandra and Nursalim, Yohanes and Parker, Kate and Lawrence, Ben and Print, Cristin G and Blenkiron, Cherie
Journal: Biopreservation and biobanking (2020)
Protein Quantitation and Analysis of Purity.
Authors: Campion, Eva M and Loughran, Sinéad T and Walls, Dermot
Journal: Methods in molecular biology (Clifton, N.J.) (2017): 225-255
Proteomic Approach for Extracting Cytoplasmic Proteins from Streptococcus sanguinis using Mass Spectrometry.
Authors: El-Rami, Fadi and Nelson, Kristina and Xu, Ping
Journal: Journal of molecular biology research (2017): 50-57
[iTRAQ technology combined with 2D-LC-MS/MS to analyze effect of Coptidis Rhizoma on cytochrome P450 isoenzyme expression].
Authors: Yang, Xin and Wang, Qiu-Hong and Wang, Meng and Wang, Yue and Yang, Bing-You and Kuang, Hai-Xue
Journal: Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica (2016): 731-736
Total protein quantitation using the bicinchoninic acid assay and gradient elution moving boundary electrophoresis.
Authors: Kralj, Jason G and Munson, Matthew S and Ross, David
Journal: Electrophoresis (2014): 1887-92
Page updated on September 24, 2024

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Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501

Platform

Absorbance microplate reader

Absorbance562 nm
Recommended plateClear bottom

Components

. BSA dose responses were measured with Amplite® Colorimetric BCA Protein Quantitation Assay Kit using a clear bottom 96-well plate.
. BSA dose responses were measured with Amplite® Colorimetric BCA Protein Quantitation Assay Kit using a clear bottom 96-well plate.
. BSA dose responses were measured with Amplite® Colorimetric BCA Protein Quantitation Assay Kit using a clear bottom 96-well plate.