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.
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 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 (Product # 26025). This reagent will detect amino groups on small peptides by fluorescence.
Do I need to make a standard curve each time I perform the 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.
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. 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, Pierce recommends the use of new plates and glassware that has been thoroughly cleaned and rinsed before use.
How can interfering substances be eliminated from the protein sample?
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). See our Tech Tips section for specific procedures.
What is the detection range for the BCA Protein Assay?
For the standard protocol, the detection range is 20-2,000µg/ml. For the enhanced test tube protocol, the detection range is 5-250µg/ml. The Micro BCA Protein Assay Reagent will detect 0.5-20µg/ml of protein in the test tube assay and 1-20µg/ml for the microplate assay.
What is the sample to working reagent ratio?
For the Microplate Protocol the sample to working reagent (WR) ratio is 1:8 (25µl sample plus 200 ul WR). If the sample amount is limited, a 1:20 ratio may be used (10 ul sample); in this case, however, the detection range of the assay will be limited to 125-2,000µg/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 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.
Which Pierce protein assay is the best or more 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. Coomassie Plus Reagent (Product # 23236) is compatible with reducing sugars, is more sensitive and is faster and easier to use.
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