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Protein Analysis and Purification Benchtop Techniques

:	Ian M. Rosenberg
:	Protein Analysis and Purification Benchtop Techniques
:	Birkhäuser Basel
:	9780817644123
:	2
:	CHF 139.30
:

:	Mikrobiologie
:	English

:	520
:	Wasserzeichen
:	PC/MAC/eReader/Tablet
:	PDF

How one goes about analyzing proteins is a constantly evolving ?eld that is no longer solely the domain of the protein biochemist. Inves- gators from diverse disciplines ?nd themselves with the unanticipated task of identifying and analyzing a protein and studying its physical properties and biochemical interactions. In most cases, the ultimate goal remains understanding the role(s) that the target protein is playing in cellular physiology. It was my intention that this manual would make the initial steps in the discovery process less time consuming and less intimidating. This book is not meant to be read from cover to cover. The expanded Table of Contents and the index should help locate what you are seeking. My aim was to provide practically oriented information that will assist the experimentalist in benchtop problem solving. The appendices are ?lled with diverse information gleaned from catalogs, handbooks, and manuals that are presented in a distilled fashion designed to save trips to the library and calls to technical service representatives. The user is encouraged to expand on the tables and charts to ?t individual experimental situations. This second edition pays homage to the computer explosion and the various genome projects that have revolutionized how benchtop scienti?c research is performed. Bioinformatics and In silico science are here to stay. However, the second edition still includes recipes for preparing buffers and methods for lysing cells.

	Preface	6
	Acknowledgments	7
	Contents	8
	An Overview of This Manual	24
	Introduction	24
	Kits, Cores, and Computers	26
	How to Use This Book	26
	Basic Laboratory Equipment	27
	Laboratory Automation	28
	Beyond Protein Analysis and Purification	29
	Protein Structure	31
	Introduction	31
	A. The Amino Acids	31
	B. The Four Levels of Protein Structure	34
	C. Chemical Characteristics of Proteins	40
	Tracking the Target Protein	47
	Introduction	47
	A. Labeling Cells and Proteins	49
	Metabolic Labeling Adherent Cells	51
	Metabolic Labeling Cells Growing in Suspension	51
	Pulse-Chase Labeling	52
	Lactoperoxidase Labeling Cell Surface Proteins	54
	Labeling Surface Proteins with IODO- GEN ®	55
	Non-radioactive Biotinylation of Cell Surface Proteins	56
	Domain-Selective Biotinylation and Streptavidin- Agarose Precipitation	57
	Labeling Isolated Proteins with Chloramine T	59
	B. Lysis: Preparation of the Cell Free Extract	60
	Lysis of Cells in Suspension ( Continuation of Protocol 3.2)	61
	Lysis of Adherent Cells ( Continuation of Protocol 3.1)	62
	C. Principles of Immunoprecipitation	62
	Immunoprecipitation	65
	Sequential Immunoprecipitation: Dissociation and Reimmunoprecipitation of Immune Complexes	67
	Eliminating Interfering Immunoglobulin Bands During IP- Western Detection: Analysis of the Immunoprecipitate under Non- Reducing Conditions	69
	Nondenaturing Immunoprecipitation	70
	D. Additional Methods to Identify Associated Proteins	70
	Preparation of Sucrose Gradients	71
	Cross-Linking Proteins Added to Cells: Analysis of Receptor- Ligand Interaction	78
	Cross-Linking Proteins in Solution	78
	Cross-Linking Extraneously Added Ligand to Cells	79
	Cross-Linking Proteins in Solution Using the Homobifunctional Reagent Dithiobis ( succinimidyl propionate) ( DSP)	80
	Electrophoretic Techniques	86
	Introduction to Polyacrylamide Gel Electrophoresis ( PAGE)	86
	A. Preparation of SDS-Polyacrylamide Gels	89
	Assembling the Plates	89
	Casting the Separating Gel	90
	Casting the Stacking Gel	92
	Gradient Gels	93
	Sample Preparation	95
	Running the Gel: Attaching the Gel Cassette to the Apparatus and Loading the Samples	96
	Drying the Gel	99
	Separation of Low Molecular Weight Proteins by Tricine- SDS- PAGE ( TSDS- PAGE)	100
	B. 2-Dimensional (2-D) Gel Systems	102
	Preparation of the Sample for Isoelectric Focusing	106
	Preparation and Running of Isoelectric Focusing Tube Gels	107
	Equilibration of the First-Dimension Gel or Strip	109
	Measuring the pH of the Gel Slices	110
	Nonequilibrium pH Gradient Electrophoresis ( NEPHGE)	112
	2-D Gels The Second Dimension: SDS- PAGE	113
	Labeling Proteins with Cyanine Dyes ( Cy3 and Cy5)	114
	Nonreducing-Reducing 2- D Gels	115
	C. Detection of Protein Bands in Polyacrylamide Gels	118
	Staining and Destaining the Gel with Coomassie Blue	119
	Coomassie Staining Using GelCode® Blue	121
	Staining Gels with SYPRO® Ruby	121
	Silver Staining	123
	Reversible Negative Staining of Proteins in Gels with Imidazole and Zinc Salts	124
	Molecular Weight Determination by SDS- PAGE	126
	D. Recovery of Proteins from the Gel	128
	Excising the Protein Band from the Dried Gel	128
	Extracting the Target Protein from the Dried Gel	129
	E. Identification of Enzyme Activity in Polyacrylamide Gels	129
	Localization of Proteases: Copolymerization of Substrate in the Separating Gel	130
	Identification of Protease Inhibitors: Reverse Zymography	131
	Locating the Enzyme Activity: Reacting the Gel with Substrate Solution after Electrophoresis	132
	Detection of b glucuronidase Activity in Polyacrylamide Gels	133
	Gel Shifts	135
	Getting Started with Protein Purification	141
	Introduction	141
	A. Making a Cell Free Extract	143
	Nuclear Extracts	149
	Total Lymphocyte Extract	150
	Subcellular Fractionation	150
	B. Protein Quantitation	151
	Bradford Standard Assay	153
	Bradford Microassay	155
	Protein Determination Using Bicinchoninic Acid ( BCA)	156
	Compatible Substances for the BCA Protein Assay	157
	Incompatible Substances	157
	NanoOrange® Protein Quantitation Assay: A Fluorescence- Based Assay of Proteins in Solution	158
	C. Manipulating Proteins in Solution	159
	Recovery of Protein by Ammonium Sulfate Precipitation	161
	Preparation of Dialysis Tubing	164
	D. Precipitation Techniques	166
	Salting Out with Ammonium Sulfate	166
	Precipitation with Acetone	168
	PEG Precipitation	169
	Removal of PEG from Precipitated Proteins	170
	Recovery of Protein from Dilute Solutions by Methanol Chloroform Precipitation	171
	Recovery of Protein by Trichloroacetic Acid ( TCA) Precipitation	172
	Concentration of Proteins by Acetone Precipitation	173
	Membrane Proteins	176
	Introduction	176
	A. Peripheral Membrane Proteins	177
	Alkali Extraction	179
	High pH Membrane Fractionation	180
	B. Integral Membrane Proteins	181
	Butanol Extraction	182
	Single-Phase Butanol Extraction	182
	C. Detergents	183
	Differential Detergent Solubilization	192
	Solubilization Trial	194
	Transfer and Detection of Proteins on Membrane Supports	200
	Introduction	200
	A. Transfer of Proteins to Membrane Supports	200
	Transfer of Proteins to Nitrocellulose or Polyvinylidene Difluoride	201
	Enhanced Capture of Small Histidine Containing Polypeptides on Membranes in the Presence of ZnCl2	204
	Dot Blots	205
	Thin-Layer Chromatography Blotting	205
	B. Staining the Blot	206
	Total Protein Staining with India Ink	206
	Reversibl