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.
This text introduces the basic methodological tools to measure inositol lipids and phosphates and also describes new approaches that have become available in the last ten years, including g RNA-silencing and the protocols involving inositol pyrophosphates.
Table of ContentsPrefaceContributors1. The Role of Inositol and the Principals of Labelling, Extraction, and Analysis of Inositides in Mammalian CellsChristopher J. Barker and Per-Olof Berggren2. HPLC Separation of Inositol PolyphosphatesChristopher J. Barker, Christopher Illies, and Per-Olof Berggren3. Molecular Manipulation and Analysis of Inositol Phosphate and Pyrophosphate Levels in Mammalian CellsJames C. Otto and John D. York4. A Femtomole-Sensitivity Mass Assay for Inositol HexakisphosphateAndrew J. Letcher, Michael J. Schell, and Robin F. Irvine5. Synthesis of InsP7 by the Inositol Hexakisphosphate Kinase 1 (IP6K1)Cristina Azevedo, Adam Burton, Matthew Bennett, Sara Maria Nancy Onnebo, and Adolfo Saiardi6. Protein Pyrophosphorylation by Diphosphoinositol Pentakisphosphate (InsP7)J. Kent Werner, Jr., Traci Speed, and Rashna Bhandari7. Synthesis and Non-Radioactive Micro-Analysis of Diphosphoinositol Phosphates by HPLC with Post-Column ComplexometryHongying Lin, Karsten Lindner, and Georg W. Mayr8. Diphosphosinositol Polyphosphates and Energy Metabolism: Assay for ATP/ADP RatioAndreas Nagel, Christopher J. Barker, Per-Olof Berggren, and Christopher Illies9. Isolation of Inositol 1,4,5-Trisphosphate Receptor-Associating Proteins and Selective Knockdown Using RNA InterferenceAkihiro Mizutani, Kastuhiro Kawaai, Chihiro Hisatsune, Hideaki Ando, Takayuki Michikawa, and Katsuhiko Mikoshiba10. Phosphoinositide-Specific Phospholipase C b1 Signal Transduction in the NucleusRoberta Fiume, Gabriella Teti, Irene Faenza, and Lucio Cocco11. Methods to Assess Changes in the Pattern of Nuclear PhosphoinositidesNullin Divecha12. Measurement of Phosphoinositide 3-Kinase Products in Cultured Mammalian Cells by HPLCFrank T. Cooke13. Phosphoinositide Analysis by Liquid Chromatography-Mass SpectrometryTrevor R. Pettitt14. Imaging Phosphoinositide Dynamics in Living CellsAnne Wuttke, Olof Idevall-Hagren, and Anders Tengholm
