Methods in Membrane Biology: Volume 9

ISBN-13: 9781461340386 / Angielski / Miękka / 2011 / 390 str.

Edward Korn

Opis

Many of the methods now in general use in membrane biology, and not already discussed in satisfactory detail elsewhere, have been covered in the eight previously published volumes of this series. Much of this ninth volume is occupied by one authoritative chapter, an unusually thorough and critical review of a relatively new and highly specialized technology that has gained rapid acceptance: immunofluorescence and immunoelectron microscopy. These are powerful experimental tools applicable in fields much broader than membrane research and employing methods drawn from widely diverse disciplines such as organic chemistry, protein chemistry, immunology, and fluorescence and electron microscopy. The temptation to use these super ficially, and deceptively, simple but fundamentally complex methods un critically is almost overwhelming. The chapter by de Petris, a pioneer in the field, is as necessary as it is rigorous, and it should long be the standard in this area of research. The second chapter in this volume is a more specialized review by Matus of the procedures for the preparation and characterization of the highly differentiated junctional regions of brain plasma membranes. These methods are central to the rapidly growing field of neurobiochemistry membrane biochemistry at perhaps its most intricate."

Spis treści

1 Immunoelectron Microscopy and Immunofluorescence in Membrane Biology.- 1. Introduction.- 2. Preparation of Antibodies and Other Ligands.- 2.1. The Antibody Molecule.- 2.2. Antisera.- 2.3. Purification of Immunoglobulin and Antibody.- 2.4. Monovalent Antibodies.- 2.5. Hybrid Antibodies.- 2.6. Other Ligands.- 3. Immunoelectron Microscopy (IEM).- 3.1. IEM Techniques.- 3.2. IEM Markers.- 3.3. General Membrane Labeling Methods.- 3.4. Preparation of Antibody-Marker Conjugates.- 3.5. Procedures for Labeling Cells and Cellular Membranes.- 3.6. Problems of Interpretation of the Pattern of Labeling on Biological Membranes.- 4. Immunofluorescence.- 4.1. Introduction.- 4.2. Outline of the Technique. Fluorescent markers.- 4.3 Applications and Interpretation. Comparison with IEM.- 5. Concluding Remarks.- 6. References.- 2 Synaptic Membranes and Junctions from Brain.- 1. Introduction.- 1.1. Criteria for Identifying Synaptic Components in Subcellular Fractions.- 2. Subcellular Fractionation Methods.- 2.1. Isolation of Synaptosomal Plasma Membrane.- 2.2. Isolation of Synaptic Junction-Enriched Subfractions.- 2.3. Purity of the Isolated Fractions.- 3. Properties of Isolated Membranes and Junctions.- 3.1. Ultrastructure.- 3.2. Protein Components.- 3.3. Prospects for Further Neurochemical Study.- 4. References.- 3 High-Sensitivity Differential Scanning Calorimetry in the Study of Biomembranes and Related Model Systems.- 1. Introduction.- 2. Differential Scanning Calorimetry.- 2.1. General Description.- 2.2. The Privalov Calorimeter.- 2.3. Fundamental Principles of DSC.- 2.4. Calorimetric Detectability of Equilibrium Processes.- 3. Applications of High-Sensitivity DSC.- 3.1. Pure Lipids.- 3.2. Lipid Mixtures.- 3.3. Biological Membranes.- 4. Conclusion.- 5. References.- 4 Paramagnetic Hydrophilic Probes in NMR Investigations of Membrane Systems.- 1. Introduction.- 2. Interaction of Paramagnetic Ions with Phospholipid Bilayers.- 2.1. Nature of the Interaction.- 2.2. Identification of Binding Sites.- 2.3. Number of Binding Sites.- 2.4. Lifetime of the Complex.- 3. Changes in the NMR Spectra of Phospholipid Vesicles Caused by Paramagnetic Ions. Separation of Signals from Inward- and Outward-Facing Molecules.- 3.1. Relaxation Probes.- 3.2. Shift Probes.- 4. Environmental Factors Affecting the Paramagnetic Changes of the N(CH3)3 Proton Resonance.- 4.1. Metal-to-Phospholipid Ratio.- 4.2. Temperature.- 4.3. Acidity of the Medium.- 4.4. Foreign Ions.- 4.5. Lipid Environment.- 5. Applications.- 5.1. Stability and Ion Permeability of Sonicated Liposomes.- 5.2. Size Determination of Sonicated Liposomes.- 5.3. Packing Differences of Phospholipid Molecules on the Inner and Outer Surfaces of the Vesicle Bilayer.- 5.4. Outside-Inside Distribution of Lipids in Sonicated Liposomes of Mixed Composition.- 5.5. Intermembrane Phospholipid Exchange.- 5.6. Structure of Serum Lipoproteins.- 5.7. Miscellaneous.- 6. References.- 5 Membrane Mutants of Mammalian Cells in Culture.- 1. Introduction.- 2. Altered Permeability.- 2.1. Nucleoside Transport.- 2.2. Amino Acid Transport.- 2.3. Methotrexate Resistance.- 2.4. Colchicine Resistance.- 2.5. Resistance to Actinomycin D and Puromycin.- 3. Altered Response to Membrane-Specific Agents.- 3.1. Ouabain Resistance.- 3.2. Lectin Resistance.- 3.3. Resistance to Diphtheria Toxin.- 3.4. Resistance to Antisera.- 3.5. Altered Adhesive Properties.- 4. Remarks Concerning Experimental Strategies.- 4.1. Cell Type.- 4.2. Cloning and Culturing.- 4.3. Single versus Complex Selections.- 4.4. Revertants.- 4.5. Hybrids.- 5. Concluding Remarks.- 6. References.