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The Bradford protein assay ... Compute the extinction coefficient and calculate the concentrations of the unknown samples. Procedure (Micro Assay, 1-10 μg protein/mL)
In biochemistry, the molar absorption coefficient of a protein at 280 nm depends almost exclusively on the number of aromatic residues, particularly tryptophan, and can be predicted from the sequence of amino acids. [6] Similarly, the molar absorption coefficient of nucleic acids at 260 nm can be predicted given the nucleotide sequence.
Extinction coefficient refers to several different measures of the absorption of light in a medium: Attenuation coefficient , sometimes called "extinction coefficient" in meteorology or climatology Mass extinction coefficient , how strongly a substance absorbs light at a given wavelength, per mass density
At a wavelength of 260 nm, the average extinction coefficient for double-stranded DNA is 0.020 (μg/mL) −1 cm −1, for single-stranded DNA it is 0.027 (μg/mL) −1 cm −1, for single-stranded RNA it is 0.025 (μg/mL) −1 cm −1 and for short single-stranded oligonucleotides it is dependent on the length and base composition.
Thus a scattering coefficient μ s and an absorption coefficient μ a can be combined into a total extinction coefficient μ = μ s + μ a. [ 6 ] Importantly, Beer also seems to have conceptualized his result in terms of a given thickness' opacity, writing "If λ is the coefficient (fraction) of diminution, then this coefficient (fraction) will ...
Variable pathlength techniques can be applied in any situation where Beer's law can be applied. It provides an analytical method that averages out minor variations in sample preparation consistency. It also provides a means to calculate concentrations without calibrations curves or serial dilution of samples.
This reaction is rapid and stoichiometric, with the addition of one mole of thiol releasing one mole of TNB. The TNB 2− is quantified in a spectrophotometer by measuring the absorbance of visible light at 412 nm, using an extinction coefficient of 14,150 M −1 cm −1 for dilute buffer solutions, [4] [5] and a coefficient of 13,700 M −1 cm −1 for high salt concentrations, such as 6 M ...
The photophysical properties of the FbFPs are determined by the chromophore itself and its chemical surrounding in the protein. The extinction coefficient (ε) is around 14.200 M −1 cm −1 at 450 nm for all described FbFPs, which is slightly higher than that of free FMN (ε = 12.200 M −1 cm −1 [10]).