How to prepare BCA standard?
The BCA Working Reagent is prepared by mixing 50 parts of Reagent A with 1 part of Reagent B. Mix the BCA Working Reagent until it is light green in color. Store Reagents A and B at room temperature. Reagent A, without Reagent B added, is stable for at least one year at room temperature in a closed container.
How to prepare BCA assay?
Assay
- Prepare samples containing 0.2 to 50 micrograms protein in microliters.
- Add 1 ml SWR to each 20 microliters sample and mix. Incubate 30 min. at 60 degrees C.
- Cool the samples and read at 562 nm. Color will be stable for at least one hour.
How does the Bradford reagent work?
How the Bradford Protein Assay Works. The Bradford protein assay is a time-tested colorimetric assay. When the Bradford reagent (acidified Coomassie Brilliant Blue G-250) binds to proteins, the dye undergoes a color change in the visible spectrum, with the absorbance maximum moving from 470 to 595 nm.
What is BCA chemical?
The BCA protein assay is used to quantify total protein in a biological sample. BCA stands for Bicinchoninic acid, which is the key reagent used to produce a colored product. The purple colored product is analyzed in reference to a standard curve in order to quantify protein concentration.
How is BCA protein concentration calculated?
Y= mX+C (eg Y=0.0545X+0.218). Here X is the unknown concentration which you need to find. Y value is the absorbance that you got for your unknown sample. Therefore X= (Y-C)/m. i.e X= (Y-0.218)/0.0545.
What is BCA protein assay?
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.
How BCA calculate protein concentration?
What is a BCA protein assay kit?
Pierce BCA Protein Assay Kit is a two-component, high-precision, detergent-compatible protein assay for determination of protein concentration. Pierce BCA reagents provide accurate determination of protein concentration with most sample types encountered in protein research.
What does Bradford assay tell you?
The Bradford protein assay is used to measure the concentration of total protein in a sample. The principle of this assay is that the binding of protein molecules to Coomassie dye under acidic conditions results in a color change from brown to blue.
How does Bradford assay determine protein concentration?
The Bradford assay is a quick and fairly sensitive method for measuring the concentrations of proteins. It is based on the shift in absorbance maximum of Coomassie Brilliant Blue G-250 dye from 465 to 595 nm following binding to denatured proteins in solution.
How does BCA detect protein?
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.
How do you quantify protein concentration?
The simplest and most direct assay method for protein concentration determination in solution is to measure the absorbance at 280 nm (UV range). Amino acids containing aromatic side chains (i.e., tyrosine, tryptophan and phenylalanine) exhibit strong UV-light absorption.
How does BCA assay calculate protein concentration?
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 does a BCA assay detect proteins?
Like the conventional BCA assay, the Pierce Rapid Gold BCA Protein assay involves the reduction of copper by proteins in an alkaline medium (biuret reaction) to produce sensitive and selective colorimetric detection by a new copper chelator.
Why are proteins measured at 280 nm?
UV-vis / A 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).
How do you calculate protein concentration in mg mL?
Use the following formula to roughly estimate protein concentration. Path length for most spectrometers is 1 cm. Concentration (mg/ml) = Absorbance at 280 nm divided by path length (cm.) Pure protein of known absorbance coefficient.
What is the purpose of BCA protein assay?
BCA (Bicinchoninic Acid) Protein Assay. 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.
How do you calculate protein concentration in Bradford assay?
To calculate the concentration of the undiluted, unknown sample, simply multiply by the dilution factor. So, 0.5 x 10= 5mg/ml.
What is R2 value in Bradford assay?
An R2 value upto 0.97 for linear regression is acceptable for Bradford reagent. As Achim suggested, the buffer could play an important role in estimation of the protein. Be sure that your buffer doesn’t contain any detergent.