Inositol Phosphates and Lipids: Methods and Protocols

ISBN-13: 9781617796890 / Angielski / Miękka / 2012 / 230 str.

Christopher J. Barker

Opis

Inositol, in its native or lipid derived forms, serves as a master building block which, when phosphorylated, leads to the construction of more than 30 unique isomeric forms that are employed in vital but diverse regulatory roles in cells. In Inositol Phosphates and Lipids: Methods and Protocols, expert researchers introduce the basic methodological tools to measure inositol lipids and phosphates and also describe new approaches that have become available in the last 10 years, including RNA-silencing and the use of fluorescently labeled PH-domains to measure inositides in real-time in live cells, new sensitive methods to measure mass of both phosphates and lipids, as well as protocols involving inositol pyrophosphates. Written in the highly successful Methods in Molecular Biology(TM) series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes sections, detailing tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Inositol Phosphates and Lipids: Methods and Protocols compiles many of the techniques that underscore phosphorylated inositol cell biology in one convenient guide.

Spis treści

Table of Contents Preface Contributors 1. The Role of Inositol and the Principals of Labelling, Extraction, and Analysis of Inositides in Mammalian Cells Christopher J. Barker and Per-Olof Berggren 2. HPLC Separation of Inositol Polyphosphates Christopher J. Barker, Christopher Illies, and Per-Olof Berggren 3. Molecular Manipulation and Analysis of Inositol Phosphate and Pyrophosphate Levels in Mammalian Cells James C. Otto and John D. York 4. A Femtomole-Sensitivity Mass Assay for Inositol Hexakisphosphate Andrew J. Letcher, Michael J. Schell, and Robin F. Irvine 5. Synthesis of InsP7 by the Inositol Hexakisphosphate Kinase 1 (IP6K1) Cristina Azevedo, Adam Burton, Matthew Bennett, Sara Maria Nancy Onnebo, and Adolfo Saiardi 6. Protein Pyrophosphorylation by Diphosphoinositol Pentakisphosphate (InsP7) J. Kent Werner, Jr., Traci Speed, and Rashna Bhandari 7. Synthesis and Non-Radioactive Micro-Analysis of Diphosphoinositol Phosphates by HPLC with Post-Column Complexometry Hongying Lin, Karsten Lindner, and Georg W. Mayr 8. Diphosphosinositol Polyphosphates and Energy Metabolism: Assay for ATP/ADP Ratio Andreas Nagel, Christopher J. Barker, Per-Olof Berggren, and Christopher Illies 9. Isolation of Inositol 1,4,5-Trisphosphate Receptor-Associating Proteins and Selective Knockdown Using RNA Interference Akihiro Mizutani, Kastuhiro Kawaai, Chihiro Hisatsune, Hideaki Ando, Takayuki Michikawa, and Katsuhiko Mikoshiba 10. Phosphoinositide-Specific Phospholipase C b1 Signal Transduction in the Nucleus Roberta Fiume, Gabriella Teti, Irene Faenza, and Lucio Cocco 11. Methods to Assess Changes in the Pattern of Nuclear Phosphoinositides Nullin Divecha 12. Measurement of Phosphoinositide 3-Kinase Products in Cultured Mammalian Cells by HPLC Frank T. Cooke 13. Phosphoinositide Analysis by Liquid Chromatography-Mass Spectrometry Trevor R. Pettitt 14. Imaging Phosphoinositide Dynamics in Living Cells Anne Wuttke, Olof Idevall-Hagren, and Anders Tengholm

Blurb

Inositol, in its native or lipid derived forms, serves as a master building block which, when phosphorylated, leads to the construction of more than 30 unique isomeric forms that are employed in vital but diverse regulatory roles in cells. In Inositol Phosphates and Lipids: Methods and Protocols, expert researchers introduce the basic methodological tools to measure inositol lipids and phosphates and also describe new approaches that have become available in the last 10 years, including RNA-silencing and the use of fluorescently labeled PH-domains to measure inositides in real-time in live cells, new sensitive methods to measure mass of both phosphates and lipids, as well as protocols involving inositol pyrophosphates. Written in the highly successful Methods in Molecular Biology™ series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes sections, detailing tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, Inositol Phosphates and Lipids: Methods and Protocols compiles many of the techniques that underscore phosphorylated inositol cell biology in one convenient guide.