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protein purification

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protein purification

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    1. PROTEIN PURIFICATION Step 1:Crude Extract

    3. Overview OfThis Purification Making the Extract Purification Steps Ammonium sulfate precipitation (salting out) Dialysis Ion Exchange Chromatography Each of these steps is a separate lecture. Each of these steps is a separate lecture.

    4. Overview OfThis Purification Cont… Analysis and Verification Assays for specific activity Polyacrylamide gel electrophoresis Western Blotting

    5. Making The Extract Homogenize the source material Goal: get target protein into solution Source material Any biological material which contains abundant source of target protein If you’re lucky… Target protein is secreted outside the cell Most proteins found inside cell

    6. Cell Disruption Enzymatic Mechanical Grinding Homogenizing Motorized or not High pressure Sonication

    8. Enzymatic MethodBug Buster™ from Novagen

    9. French Press

    11. Sonication Uses high frequency sound waves to break open bacteria Generates heat Need ethanol ice bath Buffer is protective

    12. Avestin EmusiflexC5 Can process 160 liters

    13. Two ImportantThings Method to assay protein Method to stabilize protein during the purification

    14. Proteases Cell lysis during cell disruption releases proteases from cellular compartment (lysosomes) How do we keep them from destroying target protein? Keep cold (4°C) Some protocols add protease inhibitors

    15. Choice OfExtraction Buffer Buffering pH Based on pH stability range of protein Ionic strength Divalent cations

    16. Choice OfExtraction Buffer Reducing agents Protease inhibitors Reference and resource: Seidman and Moore

    17. After Cell Disruption Centrifuge the homogenate to create the CRUDE EXTRACT Take aliquots Store at 4°C Save the pellet (Why?)

    18. Components OfLaboratory Solutions Buffers Different pH optima Usually pH 6-8

    19. Salts Ionic strength Affects 3-D structure Affects solubility Because it affects electrostatic interactions between side chains Mg, Zn, Fe

    20. Co-enzymes NAD, etc

    21. Components OfLaboratory Solutions Divalent cations Ca, Mg Reducing agents Dithiothreitol, ß-mercaptoethanol Protease inhibitors Leupeptin PMSF Etc.

    22. To Learn More…. Protein Analysis and Purification Ian M Rosenberg Reference and resource: Seidman and Moore Many web resources

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