Lecture Handout 12 - BIOT 101 Handout 12 Protein...

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BIOT 101 Handout 12 Protein Purification and Analysis Vocabulary ry Affinity chromatography Antibodies Antigen Coenzymes Cofactors Column chromatography Conformation Electroblotting Elution Fractionation Gel-filtration chromatography Gel-permeation chromatography Ion exchange chromatography Isoelectric focusing (IEF) Isolectric point (pI) Labeling Ligand Liquid chromatography Mass spectrophotometry(MS) Matrix Nuclear magnetic resonance (NMR) Proteome Proteomics Resin Size-exculsion chromatography Sodium dodecylsulfate (SDS) Two-dimensional electrophoresis Western blotting X-ray crystallography Objectives Understand what is meant by a proteome. Understand why determining an organism’s proteome is more complex than determination of its genome. Understand the difficulties in studying proteins. Be able to describe the methods that can be applied to complex mixtures of biological molecules to determine the concentration and nature of the proteins within that mixture. Understand how various types of spectrophotometer and electrophoresis can be applied to this task. Be aware of the role that X-ray crystallography, MS and NMR play in protein analysis. Be able to describe the basic theory of column chromatography and how it can be applied both in a preparatory manner and in an analytical manner. Understand the principles of separation that allow the various types of chromatography to purify proteins. I. As you are well aware of by now, proteins play important roles as enzymes, receptors, ligands, membrane channels, etc. and thus are at the center of virtually all processes 1
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BIOT 101 Handout 12 Protein Purification and Analysis that occur in living systems. For the biotechnologist, being able to determine the nature of the proteins in a complex biological mixture (such as the growth media from a fermentation run or the products of cell lysis) along with being able to purify a protein from such a complex mixture to establish the structure and function of that protein or protein complex is central to making viable products and/or understanding biological processes. A rapidly emerging field known as proteomics concentrates on fully understanding the structure of proteins, protein function and the manner in which proteins associate with other molecules. An organisms proteome consists of all the proteins that organism is capable of producing. This part of proteomics can be derived from genomic sequencing. But proteins are expressed differentially in different cells and find different environments in different cells, consequently full understanding of an organism’s proteome requires that you understand the protein expression patterns of particular cells. These are characterized as the manner in which expressed proteins interact with other proteins, what stimulates the cell to express the proteins that it produces and the affect that the expressed proteins have on the cell. To further complicate proteomics, proteins, as compared to DNA, are relatively difficult to study in
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Lecture Handout 12 - BIOT 101 Handout 12 Protein...

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