Biomembrane Protocols

by Graham, John M.; Higgins, Joan A.

• ISBN13: 9780896032507
• ISBN10: 0896032507
• Format: Paperback
• Copyright: 1994-06-01
• Publisher: Humana Pr Inc

Summary

This second volume of protocols offers the most comprehensive set of modern analytical techniques available for studying the architecture and function of membranes. It features the application of biochemical, spectroscopic, and fluorimetric methods to the investigation of molecular topology, the dynamic aspects of membrane structure, and ion transport. Antibody technology, analysis of molecules involved in intracellular signaling, and receptor-ligand interactions are also covered.

Table of Contents

Crystallization of Membrane Proteins for X-Ray Analysis

Determination of Cell Surface Polarity by Solid-Phase Lactoperoxidase Iodination

Biochemical Methods to Determine Cell Surface Topography

Labeling of Oxidized Glycoproteins with 3H-Borohydride

Identification and Separation of Integral Membrane Proteins Using Triton X-114

Surface-Selective Labeling of Polarized Proteins on Tight Epithelia Using Sulfo-N-Hydroxysuccinimido-Biotin (S-NHS-B

Use of Antipeptide Antibodies for the Isolation and Study of Membrane Proteins

Preparation of Antibodies

Affinity-Purification of Antipeptide Antibodies

Purification of Membrane Proteins by Immunoaffinity Chromatography

Competitive ELISA for Determining Membrane Protein Topology

The Production of Monoclonal Antibodies to Membrane Proteins

Production of Hybridoma Cell Lines

Screening for Monoclonal Antibody Production

Purification of a Membrane Protein (Ca2+/Mg2+-ATPase) and Its Reconstitution into Lipid Vesicles

Measurement of Protein-Protein Interactions in Reconstituted Membrane Vesicles Using Fluorescence Spectroscopy

Measurement of Lipid-Protein Interactions in Reconstituted Membrane Vesicles Using Fluorescence Spectroscopy

Determination of the Transverse Topography of Membrane Lipids Using Enzymes and Covalent Labels as Probes

Determination of the Transverse Topography of Membrane Phospholipids Using Phospholipid Transfer Proteins as Tools

Prothrombinase Complex as a Tool to Assess Changes in Membrane Phospholipid Asymmetry

Fluorescent Glycerolipid Probes: Synthesis and Use for Examining Intracellular Lipid Trafficking

Synthesis of Fluorescent Glycerolipid Probes

Delivery of Fluorescent Glycerophospholipid Probes to Cultured Cells

Synthesis and Use of Spin-Labeled Lipids for Studies of the Transmembrane Movement of Phospholipids

Measurement of Membrane Fluidity and Membrane Fusion with Fluorescent Probes Extraction and Assay of Cyclic Nucleotides

Analysis of G-Proteins Regulating Signal Transduction Pathways

Identification of G-Proteins

GTPase Studies

Reconstitution of Second Messenger Pathways in Permeabilized Cell Preparations Using GTP Analogs and Receptor Ligands

Assay of Protein Kinases and Protein Phosphorylation

Protein Kinase-Mediated Phosphorylation Events

Measurement of Protein Kinase Expression

Measurement of Protein Kinase Activity and Translocation

Analysis of Cellular Phosphoinositides and Phosphoinositols: Extraction and Simple Analytical Procedures

Biosynthesis and Extraction of Phosphoinositides and Phosphoinositols

Separation of Phosphoinositols by Anion-Exchange Chromatography

Separation of Phosphoinositides by Thin-Layer Chromatography

Deacylation of Phospholipids and Separation of Products by Anion-Exchange Chromatography

Analysis of Cellular Phosphoinositides and Phosphoinositols by High Performance Liquid Chromatography

Sample and Standards Preparation

HPLC Analytical Methods

Cytosolic Free Calcium Measurements in Single Cells Using Calcium-Sensitive Fluorochromes

Membrane Permeabilization with Bacterial Toxins

Measurement of Ion Fluxes and pH Gradients Across Cell Membranes

Ligand Binding and Processing: The Perfused Liver as a Model System

The Binding of Protein-Ligands to Cell-Surface Receptors

Density Gradient Media

Balanced Salt Solutions

Index

Table of Contents provided by Publisher. All Rights Reserved.