BCA Protein Assay Kit

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Antigen
Detection Method
Colorimetric
Detection Range
20-2000 μg/mL
Minimum Detection Limit
20 μg/mL
Application
Biochemical Assay (BCA), Quantification (Q)
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Purpose BCA Protein Assay Kit is a ready-to-use detergent-compatible Western blot related total protein analysis reagent used for the quick determination of total protein concentration by measuring absorbance at 562 nm and comparing to a protein standard absorption vs. concentration curve, according to Smith.
Sample Type Cell Culture Homogenates, Cell Culture Lysate, Plasma, Serum
Analytical Method Quantitative
Detection Method Colorimetric
Characteristics
  • Assay Type: Protein Quantitative Measurement
  • Technique: Solution-based detection of spectral absorbance at 562 nm, Smith Protein assay
  • Form Supplied:
    • BCA Reagent A and B: ready-to-use 1X solutions
    • BSA Standard: stock solution for preparing a series of standard protein dilutions
  • Physical State of A+B Reagent mixture: Light blue liquid
  • Activity: Active in alkaline medium
  • Assays per kit:
    • Tube procedure: 50 assays
    • Microplate procedure: 500 assays
  • Compatibility with reagents:
    • Compatible with typical concentrations of most ionic and nonionic detergents
    • Incompatible with chelating agents, strong acids or bases, and reducing agents: interfere with the reduction and chelating reactions of the assay mechanism
Components
  • BCA Reagent A: 100 ml
  • BCA Reagent B: 5 ml
  • Albumin (BSA) Standards: 20 ml (2 mg/ml)
Material not included
  • Test tubes or miscroplates
  • Spectrophotometer
  • Microplate Reader
Target Name (Antigen)
Alternative Name BCA
Background The BCA Protein Assay is a detergent - compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantization of total protein. This method combines the reduction of Cu 2+ to Cu 1+ by protein in an alkaline medium (Biuret reaction) with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu 1+) by bicinchoninic acid, forming a purple-colored BCA/copper complex with absorbance maximum at 562 nm. Measurement of A562 is used to determine the concentration of protein in solutions. Protein concentrations are estimated by reference to absorbances obtained for a series of standard protein dilutions (e.g. of BSA), which are assayed alongside the unknown sample. The BCA assay involves two consecutive steps of color development, where the final product indirectly depends on the protein concentration in the solution. The first step is the chelation of copper with protein in an alkaline environment, followed by reduction of the Cu2+ (cupric) to Cu1+ (cuprous) cations forming a light blue complex. It has been shown that cysteine, cystine, tryptophan, tyrosine, and the peptide bond are able to reduce Cu2+ to Cu1+. The amount of reduction is proportional to the amount of protein present (the number of peptide bonds). In the second step of the color development reaction, bicinchoninic acid (BCA), a highly specific chromogenic reagent for Cu1+, reacts with the reduced cuprous cation that was formed in step one, producing a purple complex with strong absorbance at 562 nm (Smith 1985, Wiechelman 1988). The purple-colored reaction product results from the chelation of two molecules of BCA with one cuprous ion. The intensity of the BCA/copper purple complex is proportional to the amount of protein in the sample. Thus measuring the intensity of the absorbance at 562 nm is proportional to protein concentration. The BCA protein assay demonstrates higher tolerances towards common interfering substances, such as nonionic detergents and buffer salts, than the Lowry technique.
Application Notes To be used with: Lysates or homogenates, prepared with non-reducing lysis and extraction buffers: Mammal Tissue Protein Extraction Reagent, Mammal Cell Protein Extraction Reagent, RIPA Lysis Buffer, Mitochondria Extraction Reagent, Membrane Protein Extraction Kit, Cytoplasmic and Nuclear Extraction Kit, Bacterial Cell Protein Extraction Reagent
Comment

Studying protein-protein interactions, Measuring column fractions after affinity chromatography, Assessing protein yields in whole cell lysates, High-throughput screening of fusion proteins

Restrictions For Research Use only
Storage RT
Storage Comment Store at room temperature for one year.
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