A protein in solution can be quantitatively assayed by several spectrophotometric methods. Most methods are based on binding of a chromophore to specific amino acids or bonds in the protein. The resulting color development can be detected at some wavelength of visible light. The usable range of protein concentrations is determined by the sensitivity of the assay and the relationship between absorbance (A) and protein concentration. A plot of absorbance vs. protein concentration will be linear over a limited range of protein concentration depending on the protein and the Biuret method used.
The Biuret method is used for measuring high concentrations of protein in solution. The assay has a range from 2-8 mg protein/ml. The Biuret reagent reacts with peptide bonds to form a copper-protein complex. The color developed is measured at 540 nm The standard curve was produced by measuring the absorbance of protein solutions of known concentrations can be used to estimate the protein concentration of an unknown solution. Standard curves are generated using purified protein, free of any other proteins or interfering substances.
A linear equation is calculated from the absorbance values of 4-6 different protein standards and used to estimate protein concentration from the absorbance of the unknown The unknown concentration is in our data: