AT Lecture 8

AT Lecture 8 - BISC 330L: Biochemistry Spring Semester 2012...

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BISC 330L: Biochemistry Spring Semester 2012 USC Lecture 8: Friday, 27 January 2012 1. Begin Chapter 3 material… Biochemistry Toolbox Techniques in Protein Analysis 3 0 L
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Chapter 3 : Exploring Proteins and Proteomes Biochemistry Toolbox: Techniques in Protein Purification Gaining access to proteins Purifying proteins according to: 1. Solubility 2. Size 3. Charge 4. Binding Affinity Centrifugation Gel electrophoresis
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So you want to study Protein X… Decide on a protein of interest Develop assay for Protein X Enzyme act. Determine total [protein] in mixture Specific Act. = Enzyme act. / [protein] Goal: Increase specific activity Now we can begin the purification
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Proteins come from… CELLS Where do we get proteins from? • Therefore, a protein must be separated from the other cellular components before its function and structure can be further studied.
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Homogenate/ Cell extract Sonication How do we extract proteins from cells? • In order to release the cell’s contents we must first break (lyse) the cell membrane. This process is called homogenization . Detergent lysis Mortar/Pestle Force Now what do we do?!!!
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Fig. 3.1 Example of cell fractionation by Differential Centrifugation. (Dounce homogenizer + minced tissue in cold buffer) spin
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Density gradient ( Zonal ) centrifugation separates matters according to density . Often provides a better resolution of
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AT Lecture 8 - BISC 330L: Biochemistry Spring Semester 2012...

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