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Description
The Pierce BCA Protein Assay Kit is a high-precision, detergent-compatible protein assay for determination of protein concentration. Pierce BCA reagents provide accurate determination of protein concentration with most sample types encountered in protein research. The Pierce BCA assay can be used to assess yields in whole cell lysates, affinity-column fractions, purified proteins samples, 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 concentration accuracy.
Kits are available with or without Pierce Dilution-Free BSA Protein Standards, which are a set of seven pre-diluted BSA standards, packaged in a multichannel pipette-friendly tubestrip. The tubestrip includes a single empty tube that enables users to add their own sample buffer for the purpose of blank subtraction.
The Pierce BCA Protein Assay Kit is a high-precision, detergent-compatible protein assay for determination of protein concentration. Pierce BCA reagents provide accurate determination of protein concentration with most sample types encountered in protein research. The Pierce BCA assay can be used to assess yields in whole cell lysates, affinity-column fractions, purified proteins samples, 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 concentration accuracy.
Kit options are available that include Pierce Dilution-Free BSA Protein Standards, which are a set of seven pre-diluted BSA standards, packaged in a multichannel pipette-friendly tubestrip. The tubestrip includes a single empty tube that enables users to add their own sample buffer for the purpose of blank subtraction.
Features of the BCA Protein Assay Kit include:
- Colorimetric—estimate visually or measure with a standard spectrophotometer or plate reader at 562 nm
- Accurate—exhibits half the protein-to-protein variation observed with dye-binding methods (Bradford)
- Compatible—unaffected by typical concentrations of most ionic and non-ionic detergents
- Short assay time—30-min incubation; much easier and four times faster than classical Lowry methods
- Wide assay range—linear working range for BSA of 20 to 2000 μg/mL
- Sensitive—detect down to 5 μg/mL with enhanced protocol
- No more serial dilutions—kit options include Dilution-Free BSA Protein Standards which eliminate the need to perform serial dilutions when generating a standard curve
How the assay works
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 (BCA). 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, 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 complex is approximately 100 times more sensitive (lower limit of detection) than the pale blue color of the first reaction.
The reaction is strongly influenced by four amino acid residues (cysteine, cystine, tyrosine, and tryptophan) in the amino acid sequence of the protein. However, unlike Coomassie dye-binding methods (i.e., Bradford), the universal peptide backbone also contributes to color formation, helping to minimize variability caused by protein compositional differences.
Specifications
Specifications
| Concentration | BSA: 2 mg/mL |
| Content And Storage | Sufficient For: 250 tube assays or 2500 microplate assays • BCA Reagent A, 500 mL • BCA Reagent B, 25 mL • Albumin Standard Ampules, 2 mg/mL, 10 x 1 mL Store at room temperature |
| Assay | BCA Assay |
| Detection Method | Colorimetric |
| Product Line | Pierce |
| Product Type | Protein Quantitation Assay |
| Specificity | Not Target-Specific |
| Sufficient For | 250 Tube Assays or 2500 Microplate Assays |
| For Use With (Application) | Solution-based Detection, Absorbance |
| For Use With (Equipment) | Spectrophotometer, Microplate Reader |
| Show More |
Frequently Asked Questions (FAQs)
The choice of protein assay is dependent on preferences related to assay speed, accuracy and sensitivity, as well as interfering substances in the sample to be assayed. BCA has less protein-to-protein variation, is compatible with most detergents, and has larger working range. Pierce Bradford Plus Protein Assay Kit (Cat. No. 23236) is compatible with reducing sugars, is more sensitive and is faster and easier to use. For a comparison of different protein assays and compatible reagents, see our Tech Tip: Protein Quantitation Assay Compatibility Table (https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0068-Protein-assay-compatibility.pdf).
Reducing agents, copper chelators and solutions with very high buffering capacities will interfere with the BCA assay. Reducing agents reduce the copper and will produce a high background. Copper chelators bind the copper and prevent it from being detected by the BCA reagent. High-capacity buffers prevent the BCA from reaching its optimal alkaline pH. For a complete list of compatible substances, please refer to the BCA Protein Assay Kit product instructions.
For the Microplate Protocol the sample to working reagent (WR) ratio is 1:8 (25 µl sample plus 200 µl WR). If the sample amount is limited, a 1:20 ratio may be used (10 µl sample); in this case, however, the detection range of the assay will be limited to 125-2,000 µg/ml. For the Test Tube Procedure (Standard or Enhanced Protocols) the ratio is 1:20 (0.1 ml sample plus 2.0 ml WR). In the Micro BCA Assay Kit (for both Microplate and Test Tube Procedures), the ratio is 1:1 (1 part sample plus 1 part WR).
For the standard protocol, the detection range is 20-2,000 µg/ml. For the enhanced test tube protocol, the detection range is 5-250 µg/ml. The Micro BCA Protein Assay Reagent will detect 0.5-20 µg/ml of protein in the test tube assay and 1-20 µg/ml for the microplate assay.
There are several ways to adjust a sample to be compatible with the BCA Protein Assay: • Remove the interfering substance by dialysis or gel filtration. • If the starting protein concentration of the sample is high, dilute the sample to the point that the substance no longer interferes. • Eliminate interference by copper-chelating agents by increasing the amount of copper in the working reagent (use 4 ml or 6 ml of Reagent B/100 ml of Reagent A instead of the 2 ml of Reagent B/100 ml of Reagent A that is called for in the instructions). • Precipitate sample proteins with cold acetone or trichloroacetic acid (TCA).
Safety and Handling
- SET BCA PROTEIN ASSAY KIT, 500ML KIT
- Warning
- LONG-TERM AQUATIC HAZARD Chronic 2
- ACUTE AQUATIC Acute 1
- H400-Very toxic to aquatic life.
- H411-Toxic to aquatic life with long lasting effects.
- P273-Avoid release to the environment.
- P391-Collect spillage.
- P501-Dispose of contents/container to .
- MIXTURE LIST-Contain: Copper (II) sulfatepentahydrate
For Research Use Only.
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