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Used in more labs than any other detergent-compatible protein assay.
The Thermo Scientific Pierce BCA Protein Assay remains one of the most popular protein assay methods worldwide. The detergent-compatible method provides for accurate determination of protein concentration with most sample types encountered in protein research laboratories. The Pierce BCA Assay can be used to assess yields in whole cell lysates and affinity-column fractions, as well as to monitor protein contamination in industrial applications. Compared to most dye-binding methods, the BCA Assay is affected much less by protein compositional differences, providing greater protein-to-protein uniformity.
Highlights:
- Colorimetric method; read at 562 nm
- Compatible with most ionic and nonionic detergents
- Faster and easier than the Lowry method
- Working reagent stable for 24 hours
- Linear working range for BSA from 20-2,000 µg/ml
- Minimum detection level of 5 µg/ml with the enhanced protocol
- Adaptable to microplates
- Less protein-to-protein variation than dye-binding methods
BCA Protein Assay Applications:
- Studying protein:protein interactions
- Measuring column fractions after affinity chromatography
- Estimating percent recovery of membrane proteins from cell extracts
- High-throughput screening of fusion proteins
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Substances compatible with the Thermo Scientific BCA Protein Assay. The following is a short list of compatible substances and their concentrations that the BCA Protein Assay can tolerate. For a more complete list please refer to the instruction booklet for the product or view our Table of Compatible Substances for a comparison of substance compatibilities among several different protein assay methods.
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Substance
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Compatible Conc.
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Substance
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Compatible Conc.
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NP-40
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5.0%
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Glucose (fresh)
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10 mM
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Emulgen
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1.0%
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EDTA
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10 mM
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Hepes
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100 mM
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Sodium Chloride
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1.0 M
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DTT
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1 mM
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NaOH
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0.1 M
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Guanidine•HCl
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4.0 M
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Ammonium Sulfate
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1.5 M
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Triton X-100
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5.0%
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Sodium Acetate, pH 5.5
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200 mM
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Octyl-ß-Glucoside
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5.0%
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SDS
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5.0%
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Urea
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3.0 M
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Brij-35
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5.0%
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Sucrose (fresh)
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40%
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Lubrol
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1.0%
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Glycine, pH 2.8
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100 mM
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CHAPS
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5.0%
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Please refer to the product instructions for special notes and definitions about compatibility tolerances.
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Time comparison of BCA Protein Assay Reagent vs. Lowry Protein Assay.
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BCA Protein Assay Reagent
1. Mix reagents 1 minute
2. Add sample and incubate 30 minutes
3. Read at 562 nm 1 minute
Total BCA Time: 32 minutes
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Lowry Protein Assay
1. Make reagents 70 minutes
2. Add sample and incubate 20 minutes
3. Add Folin Reagent 1 minute
4. Incubate 30 minutes
5. Read at 750 nm 1 minute
Total Lowry Time: 122 minutes
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Standard curves. Typical standard curves for bovine serum albumin (BSA) and bovine gamma globulin (BGG) in the BCA Protein Assay. Kits include ampules of Albumin Standard.
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How the BCA Protein Assay Detects Protein:
The BCA Protein Assay combines the well-known reduction of Cu2+ to Cu1+ by protein in an alkaline medium with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu1+) by bicinchoninic acid. The first step is the chelation of copper with protein in an alkaline environment to form a light blue complex. In this reaction, known as the biuret reaction, peptides containing three or more amino acid residues form a colored chelate complex with cupric ions in an alkaline environment containing sodium potassium tartrate.
In the second step of the color development reaction, bicinchoninic acid (BCA) reacts with the reduced (cuprous) cation that was formed in step one. The intense purple-colored reaction product results from the chelation of two molecules of BCA with one cuprous ion. The BCA/copper complex is water-soluble and exhibits a strong linear absorbance at 562 nm with increasing protein concentrations. The BCA reagent is approximately 100 times more sensitive (lower limit of detection) than the pale blue color of the first reaction.
The reaction that leads to BCA color formation is strongly influenced by four amino acid residues (cysteine or cystine, tyrosine, and tryptophan) in the amino acid sequence of the protein. However, unlike the Coomassie dye-binding methods, the universal peptide backbone also contributes to color formation, helping to minimize variability caused by protein compositional differences.
For more information, see the article "Chemistry of Protein Assays" in the Protein Methods Library.
References:
1. Smith, P.K., et al. (1985). Anal. Biochem. 150, 76-85.
2. Sorensen, K. (1992). BioTechniques 12(2), 235-236.
3. Ju, T., et. al. (2002). J. Biol. Chem. 277, 178-186.
4. Shibuya, T., et. al. (1989). Tokyo Ika Daigaku Zasshi 47(4), 677-682.
5. Hinson, D.L. and Webber, R.J. (1988). BioTechniques 6(1), 14, 16, 19.
6. Akins, R.E. and Tuan, R.S. (1992). BioTechniques 12(4), 496-7, 499.
7. Tylianakis, P.E., et. al.(1994). Anal. Biochem. 219(2), 335-340.
8. Gates, R.E. (1991). Anal. Biochem. 196(2), 290-295.
9. Stich, T.M. (1990). Anal. Biochem. 191, 343-346.
10. Tuszynski, G.P. and Murphy, A. (1990). Anal. Biochem. 184(1), 189-191.
Related Links:
BCA Protein Assay Reagents FAQ
Protein:Protein Variation of Protein Assays
Protein Assay Selection Guide
Protein Methods Library:
Overview of Protein Assays
Chemistry of Protein Assays
Protein Assay Data Analysis
Compatible Instrumentation:
BioMate 3 UV-Vis Spectrophotometer
BioMate 5 UV-Vis Spectrophotometer
NanoDrop 2000c UV/Vis Spectrophotometer
NanoDrop 8000 UV/Vis 8 Sample Spectrophotometer
Related Products:
BSA & BGG Protein Assay Standard Sets
Micro BCA Protein Assay Kit
BCA Protein Assay - Reducing Agent Compatible
Coomassie Plus (Bradford) Protein Assay
Pierce 660 nm Protein Assay
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