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This detailed book examines experimental approaches used to investigate the regulation and function of phosphoinositides (PtdInsP), rare eukaryotic phospholipids with a broad role in biological processes such as signal transduction, cell migration and adhesion, cell growth, subcellular organization, and membrane trafficking. The combination of complementary biochemical, mass spectrometry, and imaging methods are instrumental for the detection and quantification of PtdInsP species, as well as induced dimerization methods, affinity precipitation or co-sedimentation with liposomes, protein insertion within lipid bilayers, and enzymatic assays, or through emerging methods like native mass spectrometry and microfluidics, all of which are touched upon in this volume. Written in the highly successful Methods in Molecular Biology format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratoryprotocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, Phosphoinositides: Methods and Protocols provides detailed methodology for both specialist and novice researchers on a variety of complementary methods that have been instrumental in dissecting the regulation, dynamics, and function of PtdInsPs.
List of contents
Simultaneous Detection of Phosphoinositide Lipids by Radioactive Metabolic Labeling.- Label-Free Quantification of Phosphoinositides in Drosophila by Mass Spectrometry.- Profiling of Phosphoinositide Molecular Species in Resting or Activated Human or Mouse Platelets by a LC-MS Method.- Quantification of Genetically-Encoded Lipid Biosensors.- Detection of Plasma Membrane Phosphoinositide Dynamics Using Genetically-Encoded Fluorescent Protein Probes.- Imaging the Nanoscale Distribution of Phosphoinositides in the Cell Plasma Membrane with Single-Molecule Localization Super-Resolution Microscopy.- Induced Dimerization Tools to Deplete Specific Phosphatidylinositol Phosphates.- A Real-Time Phosphatidylinositol 4-Phosphate 5-Kinase Assay Using Fluorescence Spectroscopy.- Assessing In Situ Phosphoinositide-Protein Interactions through Fluorescence Proximity Ligation Assay in Cultured Cells.- Characterization of Protein-Phospholipid/Membrane Interactions Using a "Membrane-on-a-Chip" Microfluidic System.- Exploring Phosphoinositide Binding Using Native Mass Spectrometry.- Liposome-Based Methods to Study Protein-Phosphoinositide Interaction.- Liposome-Based Methods to Study GTPases Activation by Phosphoinositides.- Liposome Co-Sedimentation and Co-Flotation Assays to Study Lipid-Protein Interactions.- Characterization of PROPPIN-Phosphoinositide Binding by Stopped-Flow Fluorescence Spectroscopy.- Fluorescence Assays to Study Membrane Penetration of Proteins.- Fluorogenic XY-69 in Lipid Vesicles for Measuring Activity of Phospholipase C Isozymes.
