What is the wavelength of BCA?
What is the wavelength of BCA?
562 nm
The BCA– copper complex is water-soluble and exhibits a strong linear absorbance at 562 nm with increasing protein concentrations. The purple color may be measured at any wavelength between 550 and 570 nm with minimal (less than 10%) loss of signal.
What is the detection range of proteins by BCA assay?
0.2–50 μg
The BCA assay method is based on the fact that the sodium salt of bicinchoninic acid reacts with the cuprous ion generated by the biuret reaction under alkaline conditions. The bicinchoninic acid cuprous complex forms a deep blue color that is read at 562 nm, and the detection range is 0.2–50 μg.
What does the BCA assay measure?
Bicinchoninic acid (BCA) assay or Smith assay is a copper-based colorimetric assay for total protein quantification. BCA rely on the formation of a Cu2 +–protein complex in a basic environment, followed by reduction of the Cu2 + to Cu+ (Smith et al., 1985).
Why is the Bradford assay measured at a wavelength of 595 nm?
The Bradford assay, a colorimetric protein assay, is based on an absorbance shift of the dye Coomassie brilliant blue G-250. When the dye binds to the protein, it causes a shift from 465 nm to 595 nm , which is why the absorbance readings are taken at 595 nm.
Is BCA assay linear?
The BCA/copper complex is water-soluble and exhibits a strong linear absorbance at 562 nm with increasing protein concentrations.
Is a BCA a colorimetric assay?
The Bicinchoninic Acid (BCA) Protein Assay is a very common colorimetric assay for sensitive detection and measurement of total protein concentration compared to a protein standard curve.
What is BCA reagent?
The BCA protein assay is used for quantitation of total protein in a sample. The principle of this method is that proteins can reduce Cu+2 to Cu+1 in an alkaline solution (the biuret reaction) and result in a purple color formation by bicinchoninic acid.
Does SDS interfere with BCA assay?
The 2-mercaptoethanol will interfere with the BCA assay, and the SDS will interfere with the Bradford assay.
Why is the wavelength of 540 nm used in the biuret test?
The biuret reaction can be used to assess the concentration of proteins because peptide bonds occur with the same frequency per amino acid in the peptide. The intensity of the color, and hence the absorption at 540 nm, is directly proportional to the protein concentration, according to the Beer–Lambert law.
What wavelength is used for the Bradford assay?
595 nm
The “Bradford Reagent” is an acidic stain which turns blue when it interacts with protein. The resulting absorbance is best determined at 595 nm.
What is a reasonable range of BSA concentrations for a Bradford assay standard curve?
You are told to create a standard curve using the Bovine Serum Albumin (BSA) that are used in a Bradford Assay, which is good for measuring protein concentrations in the range between 0.5 and 1,000 µg/ml.
What is the BCA protein assay?
The Standard BCA Protein Assay enables the measurement of protein concentrations from 10 μg/mL to 2000 μg/mL with incubation at 37°C for 30 min or room temperature for 2 hours. The BCA color formation, triggered by copper reduction, is not an endpoint reaction but adds flexibility to the assay.
What is the absorbance of BCA reagent?
The BCA/copper complex is water-soluble and exhibits a strong linear absorbance at 562 nm with increasing protein concentrations. The BCA reagent is approximately 100 times more sensitive (lower limit of detection) than the pale blue color of the first reaction.
What is the wavelength of the bcbca assay?
BCA, Coomassie, and 660 nm Assays are 562 nm, 595 nm and 660 nm respectively. Typical spectrophotometers can be set to measure at these optimal wavelengths in the visible spectrum, but many microplate readers are equipped with filter sets for specific wavelengths that differ from the absorbance maxima required by the protein assay.
What is the working range of the BSA assay?
• Assay range —linear working range for BSA of 20 to 2000 µg/mL • Sensitivity —detect down to 5 µg/mL with enhanced protocol How the assay works
