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Bio 109 Lab 4 report.pdf - Lab 4: Quantitation of Protein...

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1Lab 4: Quantitation of ProteinGOALS:To acquaint students with the Bradford test for protein quantitationTo introduce students to the basics of spectrophotometryTo introduce students to the use of a spectrophotometerTo teach students how to prepare dilutions for analysisTo teach students how to construct a standard curveTo teach students how to utilize spreadsheet software to organize,display, and analyze dataINTRODUCTIONIt is impossible to place biological material under a microscope and countthe number (quantity) of protein molecules per unit volume the way wecan count the number of cells.Therefore, something measurable that isproportional to the concentration of the substance of interest must beidentified.Beer's law states that when a solute absorbs light of a particularwavelength, the absorbance is directly proportional to the concentrationof the solute in solution. The measurement most commonly used in proteinassays is the absorbance of light. However, proteins do not absorbsufficient light to assay - by themselves.The Bradford assay uses a dye, Coomassie Brilliant Blue G-250, which wasfirst described by Dr. M. Bradford in 1976.This assay takes advantage ofthe chemical properties of the dye and the dye’s ability to interact withthe side chains, or R-groups, of specific amino acids.Coomassie G-250exists in multiple forms.As part of the Bradford solution, the dye exists in itscationic state and takes on a reddish-brown color.The peak absorptionof the dye in this state is 470 nm.When the dye binds to and interacts withamino acids in proteins, the dye is converted to a stable unprotonatedblue form, and the absorption maximum shifts from 470 nm to 595 nm.Thedye binds to proteins using three types of interactions. The primaryinteraction of the dye with proteins occurs through arginine, a very basicamino acid, which interacts with the negatively charged sulfate groupsthrough electrostatic interactions.Other weaker dye-protein interactionsinclude the interaction of the aromatic rings of Coomassie G-250 dye withthe aromatic rings of amino acids, such as tryptophan, through electronstacking interactions. Finally, the dye also weakly interacts with polar
2amino acids that have hydrophobic R-groups, such as the aromatic ringof tyrosine.The exact chemical interactions or binding properties of Coomassie G-250dye are illustrated below.
3This stable blue form of the dye is easily observed and quantified in aspectrophotometer.In accordance with Beer’s Law, there is a correlationto the amount (absorbance) of blue color and the amount of protein(solute) in the sample. The more protein, the more intense the blue color.The simplicity of the assay allows the results to be measured qualitativelyby eye, or quantitatively with a spectrophotometer.

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Term
Fall
Professor
NoProfessor
Tags
Dye, Bradford protein assay, Coomassie

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