Lowry Protein Assay- Protein Quantification | BQCkit
Lowry Protein Quantification Assay is based on Lowry method, first described in 1951. The method relies on two different reactions. The first is the formation of a copper ion complex with amide bonds, forming reduced copper in alkaline solutions. This is called a Biuret chromophore and is commonly stabilized by the addition of tartrate. The second reaction is reduction of Folin-Ciocalteu reagent (phosphomolybdate and phosphotungstate), primarily by the reduced copper-amide bond complex as well as by tyrosine, tryptohan, histidine, cystine, and cysteine residues in protein.The monovalent copper ion catalyzes the latter reaction.
The reduced Folin-Ciocalteu reagent is blue and thus detectable with a spectrophotometer in the range of 500 to 750 nm. The Biuret reaction itself is not very sensitive. Using the Folin-Ciocalteu reagent to detect reduced copper makes the Lowry assay nearly 100 times more sensitive than the Biuret reaction alone.
The reaction occurring in the Lowry method takes place in the following phases:
1. Pre-reaction of protein with alkaline Cu2+, in presence of tartrate to avoid precipitation. It is essentially identical than Biuret reaction, forming a coordination complex between copper and peptide nitrogen.
2. Reduction of the Folin-Ciocalteu reagent by the reduced copper-amide bond complex as well as by tyrosine and tryptophan residues. The reduced Folin-Ciocalteu reagent presents a dark blue color, which the user can measure colorimetrically.
The colored complex, whose composition is unknown; it has two absorption maxima at wavelengths of 560 and 680 nm. The choice of either depends on the protein concentration of the sample.