Week 3: Bradford Assay

Spectrophotometry and and beers Spectrophotometry beers law law Introduction An absorbency spectrophotometer is an instrument that measures the fraction of the incident light transmitted through a solution In other words, it is used to measure the amount of light that passes through a sample material and, by comparison to the initial intensity of light reaching the sample, they indirectly measure the amount of light absorbed by that sample Because different compounds absorb light at different wavelengths, a spectrophotometer can be used to distinguish compounds by analyzing the pattern of wavelengths absorbed by a given sample Light of a particular wavelength passes through the solution inside the curette and the amount of light transmitted or passed through the solution - {transmittance} or absorbed {absorbance} by the solution is measured by a light meter The transmittance is defined as the ratio of the light energy transmitted through the sample to the energy transmitted through the reference blank When visible light passes through a colored solution, some wavelengths are transmitted and others are absorbed d Re 750 ge an Or ow ll Ye 620 495 450 380 n ee Gr 590 e Blu 570 let Vio The optimal wavelength (Amax) for measuring absorbance is that wavelength that is most absorbed by the compound in question The light from the spectrophotometers light source does not consist of a single wavelength, but a continuous portion of the electromagnetic spectrum Beer’s law This law [Beer’s law] States that the absorbency of a light absorbing material is proportional to its concentration in solution Beer's law: A = elc A is absorbance e is the molar absorptivity or extinction coefficient of the substance l is the sample path length measured in cm (width of cuvette) usually 1 cm c is the molar concentration of the solution Bradford protein assay Bradford protein assay How the assay works When the dye is in the acidic solution with no protein present, the dye is in the cationic form. In this form the dye has a maximum absorption at 465 - 490 nm and appears reddish brown to the eye When mixed with a protein solution, coomassie brilliant blue G250 will bird to the proteins in solution and is stabilized in the anionic form' which is blue. Maximum absorption from 595 - 600 nm Coomassie brilliant blue G250 forms strong, noncovalent complexes with proteins by electrostatic interactions with amino and carbonyl groups, and by van der waals forces Here we have to create a standard curve of known protein standards and use the equation of the standard curve to estimate protein concentration of the purified lactate dehydrogenase