Phospholipid Signaling Protocols

About this book

This wide-ranging collection of state-of-the-art techniques for the assay of phospholipid and phospholipid-derived second messengers allows the identification and quantification of signal pathway activation. The assays described cover all the major phospholipases (C, D, A2), as well as sphingomyelinase and associated metabolites. Additional protocols are provided for the assay of phosphoinositide 3-, 4-, and 5-kinase activity and for the separation and identification of phospholipids, diacylglycerol and sphingolipids, as well as their phosphoinositol, choline, and fatty acid metabolites. Detailed, step-by-step instructions make this book suitable for both the newcomer and the experienced investigator.

Contents

Part I. Monitoring of Activation of Phospholipid-Derived Cell Signaling Pathways. Phosphoinositidase C Activation Assay I: Cell Labeling Stimulation and Recovery of Cellular [3H] Phosphoinositides and [3H] Phosphoinositols Ian M. Bird. Phosphoinositidase C Activation Assay II: Simple Analysis of Recovered Cellular Phosphoinositides and Phosphoinositols Ian M. Bird. Phosphoinositidase C Activation Assay III: HPLC Analysis of Cellular Phosphoinositides and Phosphoinositols Ian M. Bird. Measurement of Phosphoinositols and Phosphoinositides Using Radio High-Performance Liquid Chromatography Flow Detection Lubo Zhang and Iain L. O. Buxton. Preparation of [3H]Phosphoinositol Standards and Conversion of [3H] Phosphoinositides to [3H] Phosphoinositols Ian M. Bird. Inositol 145-Trisphosphate Mass AssayLubo Zhang. Measurement of Cellular Diacylglycerol ContentWendy Bollinger Bollag and Richard D. Griner. Phosphatidylinositol 4-Kinases: Assays and Product Analysis Rachel E. Meyers and Lewis C. Cantley. Phosphatidylinositol(345)Trisphosphate (Ptdins(345)P3) Mass Measurement Using a Radioligand Displacement AssayJeroen van der Kaay Pete J. Cullen and C. Peter Downes. Detection of Phosphatidylinositol-4-Phosphate 5-Kinase Activity Using Thin-Layer ChromatographyGregory J. Parker Joost C. Loijens and Richard A. Anderson.Determination of Phospholipase C- or Phospholipase D-Catalyzed Phosphatidylcholine Hydrolysis Michael J. O. Wakelam and Trevor R. Pettitt. Measurement of Phospholipase D ActivityWendy Bollinger Bollag. Monitoring of Phospholipase A2 Activation in Cultured Cells Using Tritiated Arachidonic Acid Sandie I. Briand Sylvie G. Bernier and Ga'tan Guillemette. Assay of Cellular Diacylglycerol and Monoacylglycerol Lipases Bryan A. Wolf. Isotopic Efflux Studies as Indices of Phospholipase Activation Jacob Vadakekalam and Stewart Metz . Extraction and Measurements of Prostanoids and Leukotrienes by Radioimmunoassays Bruno Battistini Serge Picard Pierre Borgeat and Pierre Sirois. Measurement of Prostanoids Leukotrienes and Isoprostanes by Enzyme Immunoassays Bruno Battistini Serge Picard Pierre Borgeat and Pierre Sirois. Measurements of Arachidonic Acid Metabolites Derived from the Lipoxygenase Pathways by High-Pressure Liquid Chromatography Pierre Borgeat Serge Picard Bruno Battistini and Pierre Sirois. Measurement of Sphingomyelin and Ceramide Cellular Levels After Sphingomyelinase-Mediated Sphingomyelin Hydrolysis Pino Santana Luisa F. Fanjul and C. M. Ruiz de Galarreta. Sphingomyelin and Ceramide Mass AssayPino Santana C. M. Ruiz de Galarreta and Luisa F. Fanjul . Sphingosine Kinase: Assay and Product Analysis Ana Olivera and Sarah Spiegel. Analytical Methods and Steps to Sample Preparation for Determination of Molecular Species of Fatty Acids Sanda Clejan. HPLC Analytical Methods for the Separation of Molecular Species of Fatty Acids In Diacylglycerol and Cellular Phospholipids Sanda Clejan. Analysis of Molecular Species of Cellular Sphingomyelins and Ceramides Sanda Clejan. Part II. General methods for monitoring changes in levels of key signaling pathway proteins and associated mRNA. Solubilization and Assay of Cellular and Tissue Protein Cynthia E. Shaw. Western Immunoblot Analysis Cynthia E. Shaw and Jing Zheng. Immunohistochemistry and Immunocytochemistry Jing Zheng. Extraction of Cellular and Tissue RNA Daniel S. Millican and Ian M. Bird. Size Separation and Quantification of mRNA by Northern Analysis Ian M. Bird. Preparation of Single-Stranded Antisense cDNA Probes by Asymmetric PCR Daniel S. Millican and Ian M. Bird. Optimization of a Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Mass Assay for Low-Abundance mRNA Jacqueline M. Cale Cynthia E. Shaw and Ian M. Bird.Index.

Customer Reviews