Inositol Phosphates and Lipids

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.

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.

Serves as an easily accessible reference volume for all aspects of measurement of inositol phosphates and lipids contributed by front-line researchers in the field Combines in one volume methodologies for both inositol phosphates and lipids and the key enzymes which interconnect them Covers the new field of inositol pyrophosphates as well as several established areas in the lipid and phosphate arena

Inhalt
The Role of Inositol and the Principles of Labelling, Extraction, and Analysis of Inositides in Mammalian Cells.- HPLC Separation of Inositol Polyphosphates.- Molecular Manipulation and Analysis of Inositol Phosphate and Pyrophosphate Levels in Mammalian Cells.- A Femtomole-Sensitivity Mass Assay for Inositol Hexakisphosphate.- Synthesis of InsP 7 by the Inositol Hexakisphosphate Kinase 1 (IP6K1).- Protein Pyrophosphorylation by Diphosphoinositol Pentakisphosphate (InsP 7).- Synthesis and Nonradioactive Micro-analysis of Diphosphoinositol Phosphates by HPLC with Postcolumn Complexometry.- Diphosphosinositol Polyphosphates and Energy Metabolism: Assay for ATP/ADP Ratio.- Isolation of Inositol 1,4,5-Trisphosphate Receptor-Associating Proteins and Selective Knockdown Using RNA Interference.- Phosphoinositide-Specific Phospholipase C ?1 Signal Transduction in the Nucleus.- Methods to Assess Changes in the Pattern of Nuclear Phosphoinositides.- Measurement of Phosphoinositide 3-Kinase Products in Cultured Mammalian Cells by HPLC.- Phosphoinositide Analysis by Liquid ChromatographyMass Spectrometry.- Imaging Phosphoinositide Dynamics in Living Cells.