UID:
almafu_9958128337302883
Umfang:
1 online resource (663 p.)
Ausgabe:
First edition.
Serie:
Methods in Enzymology, Volume 557
Inhalt:
Membrane Proteins - Engineering, Purification and Crystallization, a volume of Methods In Enzymology, encompasses chapters from the leading experts in the area of membrane protein biology. The chapters provide a brief overview of the topics covered and also outline step-by-step protocol for the interested audience. Illustrations and case example images are included wherever appropriate to help the readers understand the schematics and general experimental outlines. Volume of Methods In EnzymologyContains a collection of a diverse array of topics in the area of membrane protein biology rangin
Anmerkung:
Description based upon print version of record.
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Front Cover; Membrane Proteins-Engineering, Purification and Crystallization; Copyright; Contents; Contributors; Preface; Section I: Membrane Protein Engineering, Solubilization and Purification; Chapter One: Multicolor Fluorescence-Based Screening Toward Structural Analysis of Multiprotein Membrane Complexes; 1. Introduction; 1.1. Screening approaches for membrane proteins and membrane protein complexes; 1.2. Multicolor fluorescence-based screening of multiprotein membrane complexes
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1.3. The major histocompatibility complex class I peptide-loading complex: A challenge for structural biology of membrane...2. Production of Fluorescently Labeled TAP Complexes in P. pastoris; 2.1. Construct design; 2.2. Transformation; 2.3. Colony screening; 2.4. Large-scale expression; 2.5. Orthogonal purification; 3. Production of Fluorescently Labeled TAP Complexes in Mammalian Cells; 3.1. Construct design; 3.2. Transfection; 3.3. Orthogonal purification; 4. Multicolor Fluorescence-Based Screening Approaches; 4.1. MC-FSEC analysis; 4.2. In-gel fluorescence and immunoblotting
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4.3. Monitoring peptide binding to TAP4.4. Measurement of MHC I surface presentation by flow cytometry; 5. Summary; Acknowledgments; References; Chapter Two: A Novel Screening Approach for Optimal and Functional Fusion of T4 Lysozyme in GPCRs; 1. Introduction; 2. Overall Strategy; 3. Plasmids, Yeast Strains, and Media; 3.1. Yeast strains; 3.2. Plasmids; 3.3. Media; 4. Library Construction; 4.1. Generation of T4L-containing cassettes with variable lengths of flanking regions; 4.2. Insertion of cassettes containing the T4L gene into the replacement region of the receptor
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5. Expression Screening and Functional Assays6. Results for the Yeast α-Factor Receptor Ste2p; Acknowledgments; References; Chapter Three: Membrane Preparation and Solubilization; 1. Introduction; 2. Membrane Preparation; 2.1. Choice of buffer; 2.2. Cell lysis; 2.3. Membrane fractionation; 2.4. Methodology; 3. Solubilization; 3.1. Types of detergents; 3.2. Choice of detergent; 3.3. Stability in detergent; 3.4. Alternative solubilization strategy; 3.5. Presolubilization screen; 3.6. Methodology; References; Chapter Four: Amphipathic Agents for Membrane Protein Study; 1. Introduction
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2. MP Stability in Membrane Architecture3. Conventional Detergents; 3.1. Detergent classification; 3.2. Use of conventional detergents for MP study; 3.3. Limitation of conventional detergents; 4. Novel Amphipathic Systems; 4.1. Detergent properties for MP structural study; 4.2. Small amphipathic compounds; 4.2.1. Conventional detergent variants; 4.2.2. Hemifluorinated surfactants; 4.2.3. Tripod amphiphiles; 4.2.4. Facial amphiphiles; 4.2.5. Rigid hydrophobic group-bearing amphiphiles; 4.2.6. Neopentyl glycol (NG) class amphiphiles; 4.3. Peptide-based amphiphiles
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4.4. Large amphipathic systems
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English
Weitere Ausg.:
ISBN 0-12-802193-4
Weitere Ausg.:
ISBN 0-12-802183-7
Sprache:
Englisch
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