lecture9note-yeo11 - Protein Purication and Analysis...

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Protein Puri fi cation and Analysis (02/15/11) General approach to purifying proteins Protein solubility Chromatography Electrophoresis Ultracentrifugation
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•Must have sensitive method for detection. •Select a good source for the protein. a. Rich source of material. i.e. Heart mitochondria for cytochrome C b. baker’s yeast ( Saccharomyces cerevisiae ) c. E. coli •Tissue speci fi city: Brain vs. kidney vs. eye. Chickens, cows, pigs or rats are often used. Molecular cloning techniques have allowed biochemists to over-express target proteins in bacteria by isolating the gene and placing it into a host system (over-expression strain). Protein Puri fi cation
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The fi rst step - get it out of the cell Methods of solubilizing cells Cells can be lysed by hypotonic shock. Cells with high salt inside and no salt outside will swell and rupture. Bacteria outer membranes must be digested. - Gram- negative bacteria Hen egg white lysozyme digests ± (1-4) linkages in the (glycosidic bonds) of polysaccharides. Mechanical breakage – blenders, homogenizers French press - high pressure 20,000 lbs/in 2 forced through a small hole disrupts cells Ultrasound or sonication disrupts cells.
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H-bonds, ionic interactions, van der Waals interactions, and hydrophobic interactions can be disrupted. Denaturation is the process by which a protein loses its “native” or active shape or conformation. Temperature can play a role “cold labile” “heat labile” Protect against-Proteases, Inhibitors, Changes in pH Protein can be air-denatured - egg white meringue - absorption to surfaces (glass, plastic) Damaged by oxidation O 2 Heavy metals damage proteins -they bind to protein- Cu, Hg Bacterial contamination can destroy the protein Stability: proteins can denature!!
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Stabilizing Proteins Factors Effect/Consideration Proteins are isolated and kept at a pH at which they are stable/active. Proteins are often unstable at high temperatures and so are normally purified at 4°C. Must adjust pH or temperature to inactivate degradative enzymes present in the cells so that they don’t degrade the protein of interest. Add anti-proteases. Proteins are denatured by contact to air-water interfaces or by contact with glass or plastic surfaces so they are kept relatively concentrated All above conditions must be observed. Protein solutions are sometimes stored under nitrogen or argon and/or frozen at low temps. (-70 to –196 o C). pH Temperature Degradative enzymes Absorption to surfaces Long-term storage
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