Pierce™ BCA Protein Assay Kit, 1 Kit (500 mL) - 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. Nos. 23236, A55866) 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).

Find additional tips, troubleshooting help, and resources within our Protein Assays and Quantitation Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Quantitation Support Center.

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).

Find additional tips, troubleshooting help, and resources within our Protein Assays and Quantitation Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Quantitation Support Center.

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).

Find additional tips, troubleshooting help, and resources within our Protein Assays and Quantitation Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Quantitation Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Quantitation Support Center.

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).

Find additional tips, troubleshooting help, and resources within our Protein Assays and Quantitation Support Center.

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.

Find additional tips, troubleshooting help, and resources within ourProtein Assays and Quantitation Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

The maximum concentrations in the Protein quantitation assay compatibility table for the Pierce BCA Protein Assay Kit (Cat. Nos. 23225, 23227) refers to the actual concentration in the protein sample before any working reagent is added.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

NanoDrop 2000/2000c Spectrophotometers (Cat. Nos. ND-2000, ND-2000C) are compatible with most commercial BCA protein assay kits. We offer a number of BCA protein assay kits, including, but not limited to, the BCA Protein Assay Kit (Cat. No. J63283.QA), Micro BCA Protein Assay Kit (Cat. No. 23235, and Pierce BCA Protein Assay Kit (Cat. No 23225, 23227).

To identify, compare, and choose the best protein assay for your sample type and application, you can use our interactive Protein Assay Selection Guide.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

We have not tested the Pierce BCA Protein Assay Kit compatibility with OptiPrep (Iodixanol), therefore we cannot recommend using them together.

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Quantitation Support Center.

The standards can be stored overnight at room temperature or for up to a week at 4 degrees C if they are in a tightly closed container.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Quantitation Support Center.

Yes, Y-PER Yeast Protein Extraction Reagent is compatible with our Pierce BCA Protein Assay Kit. Please see our Protein assay compatibility table in this Tech Tip (https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0068-Protein-assay-compatibility.pdf).

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

You can measure the color at wavelengths between 540nm and 590 nm, although the slope for the standard curve and overall assay sensitivity is reduced.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Quantitation Support Center.

Dark purple coloring can indicate that the buffer contains a reducing agent, such as a thiol or biogenic amines. We recommend either dialyzing or diluting the sample. Alternatively, you could remove the interfering substance using Compat-Able Protein Assay Preparation Reagent Kit (Cat. No. 23215).

Find additional tips, troubleshooting help, and resources within our Protein Assays and Quantitation Support Center.

Darker samples could indicate that the protein concentration is too high, which can be corrected by diluting the sample. The sample could also contain lipids or lipoproteins. We recommend adding 2% SDS to eliminate interference from lipids. Alternatively, you could remove interfering substances using Compat-Able Protein Assay Preparation Reagent Kit (Cat. No. 23215).

Find additional tips, troubleshooting help, and resources within our Protein Assays and Quantitation Support Center.

The sample could contain a strong acid or alkaline buffer, which could alter the working reagent pH. If this occurs, we recommend dialyzing, desalting, or dilute the sample.
Alternatively, the color may have been measured at the wrong wavelength. In this case, we recommend measuring the absorbance 562 nm.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Quantitation Support Center.

The sample could contain a copper chelating agent.
We recommend trying one of the following possible solutions:
- Dialyze, desalt, or dilute the sample
- Increase the copper concentration in the working reagent (use 50: Reagent A:B)
- Remove interfering substances from the sample using the Compat-Able Protein Assay Preparation Reagent Kit (Cat. No. 23215)

Find additional tips, troubleshooting help, and resources within our Protein Assays and Quantitation Support Center.