How does Pierce 660nm Protein Assay work?
The Pierce 660-nm Protein Assay is based on the binding of a proprietary dye-metal complex to protein in acidic conditions that causes a shift in the dye’s absorption maximum, which is measured at 660 nm. The dye-metal complex is reddish-brown and changes to green upon protein binding.
What is reagent A in BCA assay?
BCA Protein Assay Reagent A is a component of the Pierce BCA Protein Assay Kit, a two-component, high-precision, detergent-compatible assay that is used for total protein concentration determination compared to a protein standard.
How is protein assay calculated?
You can use this formula to calculate the amount of protein in your sample: [ (OD of the test sample) / (OD of the standard sample) ] X concentration of the standard sample. = 4.0 mg/mL protein (in test sample).
How do you calculate protein concentration from absorbance 595?
Determine the best fit of the data to a straight line in the form of the equation “y = mx + b” where y = absorbance at 595 nm and x = protein concentration. Use this equation to calculate the concentration of the protein sample based on the measured absorbance.
How do you calculate protein concentration?
Protein concentration can be estimated by measuring the UV absorbance at 280 nm; proteins show a strong peak here due to absorbance from Tryptophan and Tyrosine residues (commonly referred to as A 280).
What is reagent A and B of BCA?
Reagents. Reagent A: 1 gm sodium bicinchoninate (BCA), 2 gm sodium carbonate, 0.16 gm sodium tartrate, 0.4 gm NaOH, and 0.95 gm sodium bicarbonate, brought to 100 ml with distilled water. Adjust the pH to 11.25 with 10 M NaOH. Reagent B: 0.4 gm cupric sulfate (5 x hydrated) in 10 ml distilled water.
Does EDTA interfere with BCA assay?
EDTA and reducing agents, such as DTT, BME, TCEP and other disulfide reducing agents, interfere with the BCA assay. The concentration of proteins can be also overestimated if proteins form soluble aggregates in solutions.
What is Thermo Scientific Pierce 660 nm protein assay?
The Thermo Scientific Pierce 660 nm Protein Assay is a ready-to-use, detergent- and reducing agent-compatible assay reagent to quickly measure (A660 nm) total protein concentration compared to a protein standard.
Which peptides can be quantified with the 660 nm protein assay?
The Thermo Scientific 660 nm Protein Assay can quantify peptides that are at least 2,500 Da if their compositions include amino acid residues that react with the dye-metal reagent (i.e., histidine, arginine, tyrosine, tryptophan and phenylalanine).
What proteins does the 660nm protein dye interact with?
The dye interacts mainly with basic residues, such as histidine, arginine and lysine and to a lesser extent, tyrosine, tryptophan and phenylalanine. What advantages does the Thermo Scientific 660nm Protein Assay have over other protein assays?
What is the assay linear range for a 660nm assay?
If a 660nm filter is not available, the assay can be measured at wavelengths from 645 to 670nm; however, the assay linear range is 25-2,000 µg/mL and occurs only when the absorbance of the dye-protein complex is measured at 660 nm.