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Protein Extraction and Gel Electrophoresis - Lab Report Example

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This essay describes a report on an experiment conducted to determine the concentration of proteins in two samples using the Bradford assay and electrophoresis. It involved three steps, that is, protein extraction, protein quantitation, and gel electrophoresis of two samples…
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Protein Extraction and Gel Electrophoresis
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"Protein Extraction and Gel Electrophoresis"

Download file to see previous pages The sodium dodecyl sulfate coats the proteins in proportion to their molecular weight and then confers the same negative electrical charge across all proteins in the sample. The rate of migration of a polypeptide in SDS-PAGE is inversely proportional to the logarithm of its molecular weight. This means that the larger the polypeptide, the slower it migrates in a gel. The molecular weight is determined by comparing the migration of protein spots to the migration of standards. Plots of log molecular weight versus the migration distance are reasonably linear. The proteins separated by SDS-PAGE are often recovered in a procedure that involves localizing the protein of interest on the gel following SDS-PAGE, eluting the protein from the gel, removing the sodium dodecyl sulfate from the eluted sample, and finally renaturing the protein for subsequent analysis. Proteins that are eluted from gels are used in varied downstream applications successfully, such as protein chemistry, determination of amino acid composition, identification of polypeptides that correspond to specific enzyme activity, and other purposes.The analysis of protein concentrations is a significant assay in biochemistry research. The Bradford assay is one of the most widely used methods to determine concentrations of protein, relative to a standard. The technique is based on the formation of a complex between proteins in solution and the dye. This assay is commended for overall use, particularly for assessing concentrations of proteins for gel electrophoresis....
The proteins separated by SDS-PAGE are often recovered in a procedure that involves localizing the protein of interest on the gel following SDS-PAGE, eluting the protein from the gel, removing the sodium dodecyl sulfate from the eluted sample, and finally renaturing the protein for subsequent analysis. Proteins that are eluted from gels are used in varied downstream applications successfully, such as protein chemistry, determination of amino acid composition, identification of polypeptides that correspond to specific enzyme activity, and other purposes. The analysis of protein concentrations is a significant assay in biochemistry research. The Bradford assay is one of the most widely used method to determine concentrations of protein, relative to a standard. The technique is based on the formation of a complex between proteins in solution and the dye, Brilliant Blue G. This assay is commended for overall use, particularly for assessing concentrations of proteins for gel electrophoresis. It is based on observations that absorbance maximum for acidic mixtures of Coomassie Brilliant Blue G-250 that do shift from 465 nm up to 595 nm at a time when protein binding occurs. The assay is effective because of the extermination coefficient of the albumin-dye complex solution is usually constant over a range of 10-fold concentration (Westermeier, Naven & Ho?pker, 2008). The dye reacts mainly with arginine residues but less with histidine, lysine, tryptophan, tyrosine, and phenylalanine residues. Seemingly, this examination is not all that perfect for acidic or basic proteins. However, it is somewhat sensitive to the bovine serum albumin, even more than most proteins, by a factor of two. Gamma globulin (IgG) is the protein standard of preference. The objective of this ...Download file to see next pagesRead More
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