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Thermo Scientific™ Pierce™ BCA Protein Assay Reagent B

Catalog No. PI23224
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PI23224 25 mL
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Catalog No. PI23224 Supplier Thermo Scientific™ Supplier No. 23224
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Description

Green-to-blue (A562nm), two-component, precise, detergent-compatible assay reagent to measure total protein concentration vs. protein standard.

Thermo Scientific™ Pierce BCA Protein Assay Kit remains one of the most popular protein quantitation methods worldwide.

Additional Information and Online Ordering

Used in more labs than any other detergent-compatible protein assay, 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 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 – estimate visually or measure with a standard spectrophotometer or plate reader (562nm)
  • Excellent uniformity – exhibits less protein-to-protein variation than dye-binding methods
  • Compatible – unaffected by typical concentrations of most ionic and nonionic detergents
  • Moderately fast – much easier and four times faster than the classical Lowry method
  • High linearity – linear working range for BSA equals 20 to 2000μg/mL
  • Sensitive – detect down to 5μg/mL with the enhanced protocol

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

Specifications

Content And Storage Store at room temperature.
Description Pierce BCA Protein Assay Reagent B
Assay BCA Assay
Detection Method Colorimetric
Product Line Pierce
Product Type Protein Quantitation Assay
Specificity Not Target-Specific
For Use With (Application) Solution-based Detection, Absorbance
For Use With (Equipment) Spectrophotometer, Microplate Reader
Quantity 25 mL

Frequently Asked Questions (FAQs)

Which Thermo Scientific protein assay is the best or the most reliable?

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).
What substances interfere with the BCA Protein Assay?

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.
What is the sample to working reagent ratio when using the BCA Protein Assay Kit?

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).

What is the detection range for the BCA Protein Assay?

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.

How can interfering substances be eliminated from the protein sample when using the BCA Protein Assay Kit?

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).

Is a specific microplate recommended for the BCA Protein Assay?

Although the brand of plate should not affect the BCA Protein Assay, assay results may be affected by well configuration. The microplate protocol is based on the use of clear, flat-bottomed plates. The recommended plates for use with the BCA Protein Assay are Pierce 96-Well Polystyrene Plates, Corner Notch (Cat. No. 15041). If these plates are not available, other clear flat-bottomed plates are acceptable as the plate surface chemistry will not alter BCA Protein Assay results. Using round-bottomed plates may affect the A(562) readings because the plate reader may not be able to align the plate within the optical path with reproducibility, which will cause variation in the absorbance from well to well. To achieve optimum results, Thermo Fisher Scientific recommends the use of new plates and glassware that have been thoroughly cleaned and rinsed before use.

Do I need to make a standard curve each time I perform the BCA Protein assay?

For optimal accuracy, make a standard curve each time a protein assay is performed. Measure standards in duplicate for the test tube assay and in triplicate for the plate assay.

Can the BCA Protein Assay Kit quantitate peptides?

Peptides smaller than 2,000 Da cannot be quantitated with the BCA assay because color formation is dependent on the protein's macromolecular structure, the number of peptide bonds, and the presence of cysteine, cystine, tryptophan and tyrosine. For peptides smaller than 2,000 Da, try using Fluoraldehyde o-Phthalaldehyde Reagent Solution (Cat. No. 26025). This reagent will detect amino groups on small peptides by fluorescence.

Other peptide quantitation assays we offer are Thermo Scientific Pierce Quantitative Colorimetric Peptide Assay (Cat. No. 23275) and Thermo Scientific Pierce Quantitative Fluorometric Peptide Assay (Cat. No. 23290).
At what wavelength is the BCA Protein Assay measured?

The maximum absorbance of the BCA-Cu+1 complex occurs at 562 nm. If a spectrophotometer or plate reader with a 562 nm filter is not available, the purple color may be measured at any wavelength between 540 nm and 590 nm. Measuring the absorbance at any wavelength other than 562 nm will result in a lower slope for the standard curve and may increase the minimum detection level for the protocol.

How does the Pierce BCA Protein Assay Kit work?

The Pierce 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.
Can you provide the shelf-life for the Pierce BCA Protein Assay Kit?

The Pierce BCA Protein Assay Kit is covered under our general 1-year warranty and is guaranteed to be fully functional for 12 months from the date of shipment, if stored as recommended (room temperature). Please see section 8.1 of our Terms & Conditions of Sale (https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/Terms-and-Conditions-of-Sale.pdf) for more details.

How do I determine the coupling efficiency of my protein to a solid support?

Immobilized protein samples can be quantified using Pierce BCA Protein Assay or Coomassie Plus Protein Assay. Please refer to this Tech Tip on how to "Quantitate Immobilized Protein"(http://tools.thermofisher.com/content/sfs/brochures/TR0009-Quant-immobilized-protein.pdf) . Alternatively, this amount can be estimated by measuring the protein concentration in solution before and after coupling. The difference in the two measurements is the amount that is bound to the support

For Research Use Only. Not for use in diagnostic procedures.