Lecture 4 Protein_Purification-handouts

Lecture 4 Protein_Purification-handouts - Chapter 3 Protein...

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Chapter 3 – Protein Purificatoin Why do we purify proteins? Proteins are purified and studied in the laboratory using a variety of techniques, often in combination. Using purified proteins, it is possible to analyze the individual proteins for their structures and properties. By mixing together purified proteins, biochemists try to determine whether a particular in vivo effect and be mimicked in vitro. Biochemists will also try to define the structure/function relationship of a protein by altering the sequence of the protein and testing the function of the mutant for comparison to the normal or wild-type protein. Protein Purification Textbook: Stryer 3.1-3.6 Outline: • Proteins have different physical properties • These properties can be used to isolate populations of specific proteins from one another Protein properties • Different solubilities • Different lengths of amino acid chain • Different charges • Different 3D shapes • Different surface features/ligand binding • Purifying proteins takes advantage of these differences to isolate a population of a protein of interest
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Protein Purification Techniques – Large Scale • Salting out • Dialysis • Gel-Filtration Chromatography • Ion-Exchange Chromatography • Affinity Chromatography • High-Pressure Liquid Chromatography Protein Purification Flowchart cell Membranes, insoluble matter Soluble matter DNA, RNA, etc. proteins 99% of all of the proteins in the cell A population of one protein of interest Darnel , Lodish, Baltimore, Molecular Cel Biology Proteins in a cell • In a cell, the protein concentration is very high (~300 mg/ml) • Other components –Membranes, DNA, RNA, sugars, structural elements • Goal: Isolating a specific protein from a cell for further study Ellis and Minton, Nature 425 , 27 - 28 (04 Sep 2003) 3 Medalia, O. et al. Science 298, 1209–1213 (2002)
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Biochemical Assays for Purification More the specific assay, more effective the purification Easier the assay, the more rapid the purification Specific activity is determine from assay and protein concentration: (units of activity/mg protein) Protein properties Different solubilities • Different lengths of amino acid chain • Different charges
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This note was uploaded on 10/20/2008 for the course BIOLCHEM 415/515 taught by Professor Ballou during the Fall '08 term at University of Michigan.

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Lecture 4 Protein_Purification-handouts - Chapter 3 Protein...

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