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lecture_8 - Protein Evolution and Analysis February 5 2003...

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Protein Evolution and Analysis February 5 2003
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Protein Assays An assay is a method of detection Specific Sensitive Convenient to use
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Enzyme-linked Immunosorbent Assay Usable in a complex mixture High sensitivity
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Electrophoresis The migration of ions in an electric field F e = qE where q is the charge E is the electric Field strength
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Electrophoresis a) F e = qE Opposing this is is the frictional force b) F f = µ v where v = velocity of migration µ is the coefficient of friction. Therefore substituting equation a) into b) qE = µ v
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Electrophoresis qE = µ v Therefore when F e = F f v= qE/µ
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Separates on charge and size pH matters as well as the pI of the protein. Can be run at several pH values depending on proteins. DNA can also be separated on agarose gels. Genomic sized DNA can also be separated but requires more sophisticated equipment .
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Paper electrophoresis
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Acrylamide gel electrophoresis
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Disc gel using a glass tube
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Polyacrylamide gel tube Electrophoretogram
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Proteins can be visualized by several methods Stained with a Dye: Coomassie blue Fluorescamine stain for fluorescence Silver staining very sensitive proteins can be labeled with radioactivity and visualized by exposure to X- ray film
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SDS-PAGE Add sodium dodecyl sulfate, a 12 carbon detergent to give a negative charge to the protein. SDS also denatures the protein and collapses into a globular ball . The proteins are separated by molecular mass
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Chromatography Analytical methods used to separate molecules. Involves a mobile and a stationary phase. Mobile phase is what the material to be separated is dissolved in. Stationary phase is a porous solid matrix which the mobile phase surrounds. Separation occurs because of the differing chemistries each molecule has with both the mobile and stationary phase.
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