Quantitative of protein Huda Hania

Methods of Quantitative of protein Method 1: protein assay based on dye binding assay Method 2: protein assay based on alkaline copper

Method 1: protein assay based on dye binding assay

BRADFORD METHOD Use of coomassie G-250 dye in a colorimetric reagent for the detection and quantitation of total protein . In the acidic environment of the reagent, protein binds to the coomassie dye. This results in a spectral shift from the reddish/brown form of the dye (absorbance maximum at 465nm) to the blue form of the dye (absorbance maximum at 610nm).

The difference between the two forms of the dye is greatest at 595nm, so that is the optimal wavelength to measure the blue color from the coomassie dye-protein complex Development of color in coomassie dye-based (Bradford) protein assays has been associated with the presence of certain basic amino acids (primarily arginine, lysine and histidine) in the protein.

Free amino acids, peptides and low molecular weight proteins do not produce color with coomassie dye reagents. (unbound) forms are green or red. Samples treated with the Bradford assay. The brown sample (lower absorbance) contains no protein, while the blue sample (higher absorbance) contains protein. The amount of protein in the second sample can be determined by comparison to a standard curve

Advantages: This assay is quick, and the reagent is not affected by the presence of reducing agents, like DTT Disadvantages: Basic conditions and detergents, such as SDS, can interfere with the dye’s ability to bind to the protein

Biuret Test

Principle: Under alkaline conditions substances containing two or more peptide bonds form a purple complex with copper salts in the reagent.  

Biuret Reagent contains: Hydrated Copper sulphate – this provides the Cu (II) ions which form the chelate complex. Cu (II) ions give the reagent its characteristic blue color. Potassium hydroxide does not participate in the reaction but provides the alkaline medium. Potassium sodium tartrate (KNaC4H4O6·4H2O) stabilizes the chelate complex

The Biuret reaction can be used to measure 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.

Conc Unknown Protein standard D.W tubes 0.5 1 0.4 0.1 2 0.8 0.2 0.3 3 1.2 4 1.6 5 6 Add 1ml of biuret reagent to each tube and mix Incubation 30 min Read at 540 nm Draw the standard curve and measure the unknown

A standard curve is a type of graph used as a quantitative research technique. Multiple samples with known properties are measured and graphed, which then allows the same properties to be determined for unknown samples by interpolation on the graph. The samples with known properties are the standards, and the graph is the standard curve. For example, a standard curve for protein concentration is often created using known concentrations of bovine serum

Reference range Reference range for total proteins is 66.6 to 81.4 g/L