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Expt3 Bradford Assay- Sample Journal Article Report F09

Expt3 Bradford Assay- Sample Journal Article Report F09 -...

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Super Writer Sr. Chem 4141-Sec 02 TA: Pleasant T. Assistant Lab Partner: Wonderful L. B. Pardtner September X, 200X Exp 3: Determination of Protein Concentration – Bradford Assay
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Summary : Protein concentration can be determined through a variety of methods, one of which is by using the Bradford assay which involves the binding of Coomassie Brilliant Blue G-250 dye to the protein under acidic conditions (where the dye is thought to bind basic and aromatic residues), causing a shift in the maximum absorption wavelength of the dye from 465 nm to 595 nm, where the absorbance of the dye at 595 nm is correlated to protein concentration. 1, 2 To determine the concentration of a solution of bovine serum albumin (BSA) of unknown concentration, a calibration curve was first constructed via serial dilutions of a standard solution of BSA whose absorbances were then analyzed after mixed with the Bradford reagent according to standard assay procedures. 1, 3 The standard curve determined in this manner followed the equation Y = 0.0004385x + 0.0125865. Dilutions were then made of the unknown sample of BSA and treated with the Bradford Reagent under standard assay procedures. 3 The absorbences of these dilutions was determined spectrophotometrically and by using the standard curve and multiplying these values by the dilution factor used the concentrations of the unknown samples was then determined. The concentration of the original unknown sample was determined to be an average of 8038 μ g/mL with a deviation of 8038 μ g/mL ± 1080 μ g/mL. It was seen that the Bradford assay is a quick and effective method for determining an unknown protein concentration, and that this method is highly sensitive, able to be effective around concentrations of about 1 μ g/mL. Introduction : The Bradford assay is a very popular protein assay method because it is simple, rapid, inexpensive and sensitive. The Bradford Assay is based on protein binding of Coomassie
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Brilliant Blue G-250 dye to protein residues in acidic solution and provides various advantages over other methods. The dye is believed to bind especially strongly to arginine residues and less strongly to histidine, lysine, tyrosine, tryptophan, and phenylalanine residues where the binding of dye to protein in acidic solution causes a shift in wavelength of maximum absorption of the dye from 465 nm to 595 nm that is linear to protein concentration. 1, 2 The absorption at 595 nm, then, is directly related to the concentration of the protein. 1, 2 Generally, a calibration curve is prepared using bovine plasma gamma globulin or bovine serum albumin as a standard. The assay requires only a single reagent, an acidic solution of Coomassie Brilliant Blue G-250 shown below: 4 After addition of dye solution to a protein sample, color development is complete in 2 minutes, and the color remains stable for up to 1 hour. With a micro assay procedure, the Bradford assay can be used to determine proteins in the range of 1 to 20 μ g. The Bradford method not only is rapid, but also has very little interference by non-protein components. The
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