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Unformatted text preview: B iochemis ry E duca ion Department of iochemistry & Molecular iology University of New Mexico BIOC 423 Int oducto y Biochemist y Methods of Protein Purification and Characterization OBJECTIVES What characteristics do biomolecules, in general, and proteins, specifically, have that can be used for separation? Be able to describe the rationale and basic chemistry (not the details) behind the various purification strategies Be able to design a purification strategy for a mixture of proteins Given a purification protocol, correctly analyze that strategy and make recommendations for improvement. What differentiates preparative techniques from analytical techniques? Be able to determine the sequence of a protein given information from Edman degradation chemistry and proteolytic and chemical fragmentation of the protein. Why is it useful to know the sequence of a protein? OUTLINE Overview of Purification What characteristics do proteins have that can be used for separation? Measuring the purification process Overview of characterization Structural Functional Separation on the basis of size/mass: Dialysis Gel Filtration Centrifugation Gel Electrophoresis Separation on the basis of charge Ion exchange chromatography Isoelectric focusing (2-D gel electrophoresis) Separation on the basis of function: Affinity Chromatography Protein Sequencing Steps in sequencing Indirect protein sequence through DNA sequence Use of protein sequence information LECTURE Overview of Purification It is often possible to learn a significant amount about biological molecules without going to the effort to purify the molecule. Many kinetic parameters and activities of enzyme inhibitors generally do not need a purified molecule. However, to make antisera, determine protein sequence and structure requires a purified molecule. In order to purify a protein there are several steps that must be followed. 1. Choose the tissue that contains the specific protein 2. Free the protein from the cell. Methods of opening up a cell include: a. Homogenization - with or without added abrasive b. Chemical denaturation of membranes - SDS, ethanol, etc.. c. Enzymatic digestion of cell walls - e.g. lysozyme and proteinase often used to "soften" bacterial cell walls d. Ultrasonic disruption e. Decompression - i.e. French press 3. Purify the molecule from the rest of the stuff. There is no one correct method to accomplish step 3 above. To accomplish purification you need to ask yourself, what characteristics do biomolecules have that we could use for separation? The answer generally, but not always, involve: Size Net Charge Affinity, i.e. what does the protein like and bind to? This is correlated to function of the protein Polarity? Solubility?...
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This note was uploaded on 04/05/2008 for the course BIOCHEM 423 taught by Professor Osgood during the Spring '08 term at New Mexico.
- Spring '08
- molecular biology