Chapter 5_Printed

Chapter 5_Printed - Chapter 5 Protein Purification and...

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Chapter 5 – Protein Purification and Characterization Techniques 1. Extracting Pure Proteins from Cells a. How do we get the proteins out of the cells? i. Homogenization – breaking open the cells 1. Grinding tissue in a blinder with a suitable buffer 2. Potter-Elvejhem Homogenizer a. A gentler technique than grinding b. A thick-walled test tube through which a tight fitting plunger is passed. The squeezing of the homogenate around the plunger breaks open cells, but leaves many of the organelles intact 3. Sonication a. Involves using sound waves to break open the cells 4. Cycles of freezing and thawing ii. Differential Centrifugation – performed after homogenization 1. 600x g: unbroken cells and nuclei 2. 15,000 x g: mitochondria 3. 100,000 x g: microsomal fraction (ribosomes and membrane fragments) iii. Salting out – after proteins are solublized 1. A crude purification based on solubility using Ammonium sulfate 2. Proteins have varying solubilities in polar and ionic compounds a. Proteins remain soluble because of their interactions with water
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b. When ammonium sulfate is added to a protein solution, some of the water is taken away from the protein to make ion-dipole bonds with the salt c. With less water available to hydrate the proteins, they begin to interact with each other through hydrophobic bonds d. At a defined amount of ammonium sulfate, a precipitate that contains contaminated proteins formed 3. The contaminated proteins are centrifuged down and discarded 4. More salt is added and a different set of proteins, which usually contains the protein of interest, precipitates a. This precipitate is collected by centrifugation and saved. 5. These preliminary techniques do not generally give a sample that is very pure, but they serve the important task of preparing the crude homogenate for the more effective procedures that follow 2. Column Chromatography a. Mobile Phase and Stationary Phase i. The mobile phase flows over the stationary material and carries the sample to be separated along with it ii. The components of the sample interact with the stationary phase to different extents iii. The differing mobilities of the components are the basis of separation
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b. Different types of Chromatography
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This note was uploaded on 03/22/2009 for the course CHEM 430 taught by Professor Redinbo during the Spring '07 term at UNC.

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Chapter 5_Printed - Chapter 5 Protein Purification and...

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