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P1603 Sigma

Phospholipid Signaling Protocols

  •  ISBN-10 0-89603-491-7

  •  ISBN-13 978-0-89603-491-4



Related Categories Biochemistry Books, Books and Software, Cell Signaling Books, Labware
publication info   I. Bird, ed., Humana Press, 1998, 400 pp., hard cover
mfr. no.   Humana


General description

This collection of state-of-the-art techniques for the assay of phospholipid and phospholid-derived second messengers allows the identification and quantitation of signal pathway activation. Techniques 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 activities and for the separation and identification of phospholipids, diacylglycerol and sphingolipids, as well as their phosphoinositol, choline and fatty acid metabolites.

Table of Contents

Part I.
Monitoring of Activation of Phospholipid-Derived Cell Signaling Pathways.
Phosphoinositidase C Activation Assay I: Cell Labeling, Stimulation, and Recovery of Cellular [3]-Phosphoinositides and [3H] Phosphoinositols,
Phosphoinositidase C Activation Assay II: Simple Analysis of Recovered Cellular Phosphoinositides and Phosphoinositols.
Phosphoinositidase C Activation Assay III: HPLC Analysis of Cellular Phosphoinositides and Phosphoinositols.
Measurement of Phosphoinositols and Phosphoinositides Using Radio High-Performance Liquid Chromatography Flow Detection,
Preparation of [3H]Phosphoinositol Standards and Conversion of [3H] Phosphoinositides to [3H] Phosphoinositols,
Inositol 1,4,5-Trisphosphate Mass Assay,
Measurement of Cellular Diacylglycerol Content.
Phosphatidylinositol 4-Kinases: Assays and Product Analysis,
Phosphatidylinositol(3,4,5)Trisphosphate (Ptdins(3,4,5)P3) Mass Measurement Using a Radioligand Displacement Assay,
Detection of Phosphatidylinositol-4-Phosphate 5-Kinase Activity Using Thin-Layer Chromatography,
Determination of Phospholipase C- or Phospholipase D-Catalyzed Phosphatidylcholine Hydrolysis,
Measurement of Phospholipase D Activity,
Monitoring of Phospholipase A2 Activation in Cultured Cells Using Tritiated Arachidonic Acid,
Assay of Cellular Diacylglycerol and Monoacylglycerol Lipases.
Isotopic Efflux Studies as Indices of Phospholipase Activation.
Extraction and Measurements of Prostanoids and Leukotrienes by Radioimmunoassays,
Measurement of Prostanoids, Leukotrienes, and Isoprostanes By Enzyme Immunoassays.
Measurements of Arachidonic Acid Metabolites Derived from the Lipoxygenase Pathways By High-Pressure Liquid Chromatography,
Measurement of Sphingomyelin and Ceramide Cellular Levels After Sphingomyelinase-Mediated Sphingomyelin Hydrolysis,
Sphingomyelin and Ceramide Mass Assay.
Sphingosine Kinase: Assay and Product Analysis,
Analytical Methods and Steps to Sample Preparation For Determination of Molecular Species of Fatty Acids,
HPLC Analytical Methods for the Separation of Molecular Species of Fatty Acids In Diacylglycerol and Cellular Phospholipids.
Analysis of Molecular Species of Cellular Sphingomyelins and Ceramides.
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.
Western Immunoblot Analysis,
Immunohistochemistry and Immunocytochemistry.
Extraction of Cellular and Tissue RNA,
Size Separation and Quantification of mRNA by Northern Analysis,
Preparation of Single-Stranded Antisense cDNA Probes by Asymmetric PCR.
Optimization of a Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Mass Assay for Low-Abundance mRNA.

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