What reagents are required in a Lowry protein quantitation assay?

Lowry Method Lowry adds phosphomolybdic/phosphotungstic acid also known as Folin-Ciocalteu reagent. This reagent interacts with the cuprous ions and the side chains of tyrosine, tryptophan, and cysteine to produce a blue-green color that can be detected between 650 nm and 750 nm.

What does the Lowry assay detect?

The Lowry protein assay is a biochemical assay for determining the total level of protein in a solution. The total protein concentration is exhibited by a color change of the sample solution in proportion to protein concentration, which can then be measured using colorimetric techniques.

How does a Lowry assay work?

The Lowry protein assay uses copper, which bonds with the peptide bonds in proteins under alkaline conditions. This forms a monovalent copper ion which can then react with the Folin reagent, which in turn can be reduced into a blue colored substance. Thus, the concentration of protein can be determined.

How do you make Lowry reagent?

The Lowry Reagent Solution is prepared by adding 40 ml of water to a bottle of Lowry Reagent, Powder (Product Code L 3540). Mix well by inverting to completely dissolve the contents. Do not shake so as to minimize foaming.

What is the basic principle of Lowry method?

The principle behind the Lowry method of determining protein concentrations lies in the reactivity of the peptide nitrogen[s] with the copper [II] ions under alkaline conditions and the subsequent reduction of the Folin- Ciocalteay phosphomolybdic phosphotungstic acid to heteropolymolybdenum blue by the copper- …

What is a BSA standard curve?

A standard curve is a plot of absorbance vs. a varying amount of some known concentration of protein. As long as the volume of the standard samples and the unknown samples are the same the final concentration of the unknown is directly calculated from the least squares line of the standard curve.

Who invented Lowry assay?

Oliver H. Lowry
Lowry assay, proposed by Oliver H. Lowry in 1951, is based on two chemical reactions (Figure 3). The first reaction is the reduction of copper ions under alkaline conditions, which forms a complex with peptide bonds (the Biuret reaction discussed above).

Why is BSA used in biuret test?

An aqueous solution of bovine serum albumin (BSA) is commonly used as a standard in testing because of its stability in testing, low cost, and is readily available as a byproduct from bovine blood.

Why is BSA used as standard?

BSA is used because of its stability to increase signal in assays, its lack of effect in many biochemical reactions, and its low cost, since large quantities of it can be readily purified from bovine blood, a byproduct of the cattle industry.

Is BSA a protein?

BSA is a protein found predominantly in the circulatory system of the cow but is also a constituent of the whey component of bovine milk.

Why is BSA used in Biuret test?

How do you create a BSA standard curve?

To create a standard curve using your BSA standards, pipette 20 μL of each standard into an eppendorf tube. Add 980 µL of the prepared dye reagent to each tube and vortex briefly. Incubate the tubes at room temperature for 5 to 50 minutes.

What kind of reagent does Modified Lowry protein assay use?

The Modified Lowry Protein Assay uses a stable reagent that replaces two unstable reagents described by Lowry. Essentially, the assay is an enhanced biuret assay involving copper chelation chemistry.

What kind of absorbance is found in Lowry assay?

Mechanism. Therefore, cysteine residues in protein probably also contribute to the absorbance seen in the Lowry assay. The result of this reaction is an intense blue molecule known as heteropolymolybdenum Blue. The concentration of the reduced Folin reagent (heteropolymolybdenum Blue) is measured by absorbance at 660 nm.

How are cell numbers normalized in Lowry assay?

The normalization of cell numbers is essential when comparing different cell types in the same assay. Normalization can normally be achieved by using a colorimetric protein assay method such as the bicinchoninic acid, the Bradford, or the Lowry protein assays.

What is the function of BCA in Lowry assay?

BCA is a weak acid composed of two carboxylated quinoline rings and serves the purpose of the Folin reagent in the Lowry assay, namely, to react with the product (Cu1 + ions) of complexes between copper ions and peptide bonds to produce a purple end product that strongly absorbs at 562 nm ( Smith et al., 1985 ).