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