chem_430_skeleton2_Notes

chem_430_skeleton2_Notes - Hogan, Chem 130 Lecture notes...

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Hogan, Chem 130 Lecture notes Chapter 5: Protein purification I. Characteristics of Proteins A. Every Protein has a unique primary sequence. Hence, every protein has several unique characteristics 1. 2. 3. 4. 5. B. As biochemists, we are interested in understanding the chemical and biological properties of proteins. Consequently, we need methods to selectively isolate proteins of interest from the rest of the cellular milieu. 1. Approx. # of proteins in an E. coli cell: 2. Approx. # of proteins in a human cell: II. Precautions A. Factors that influence protein stability: 1. 2. 3. III. Methods of cell disruption A. There are a number of ways to prepare a protein extract from prokaryotic and eukaryotic cells. 1. Mechanical methods: 2. Enzymatic methods: B. Maintaining the stability of proteins once cells have been lysed 1
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Hogan, Chem 130 Lecture notes 1. Be sure to keep your proteins happy: C. Monitoring for the presence of a protein 1. Enzyme Activity Assay: 2. Immunological: III. Protein Purification Methods 1. 2. 3. 4. A. Purification based on Solubility 1. The effects of salt concentration on solubility 2. The effects of pH on solubility 2
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Hogan, Chem 130 Lecture notes Isoelectric points of several common proteins Protein PI Protein PI Pepsin <1 Myoglobin 7.0 Ovalbumin 4.6 Hemoglobin 7.1 Tropomyosin 5.1 Ribonuclease A 9.4 Insulin 5.4 Cytochrome C 10.6 Fibrinogen 5.9 Histone 10.8 γ -Globulin 6.6 Lysozyme 11 Collagen 6.6 Salmine 12.1 B. Chromatographic Methods 1. Ion exchange chromatography: takes advantage of the intrinsic charge of a protein 2. Gel filtration (Size Exclusion) Chromatography: 3. Natural Binding Affinity: Many proteins naturally bind small molecules or substrates. 3
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Hogan, Chem 130 Lecture notes II. Visualizing Proteins: Proteins are charged; therefore, they will migrate in an electric field. Problem: Will acidic and basic proteins migrate in the same manner? A.
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This note was uploaded on 11/04/2011 for the course BIOL 430 taught by Professor Lspremulli during the Fall '08 term at UNC.

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chem_430_skeleton2_Notes - Hogan, Chem 130 Lecture notes...

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