This detailed volume explores methods that reflect the explosion of interest in the important cellular messengers known as inositol pyrophosphates. After a brief historical perspective of the analytical approaches that have contributed to the development of this research field, the book collects methods that cover isolation, detection, and measurement of inositol pyrophosphates, followed by approaches to ascertain their mechanism of action, as well as a description of the use of experimental models that have been instrumental to many fundamental discoveries. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips for troubleshooting and avoiding known pitfalls.

Authoritative and practical, Inositol Pyrophosphates: Methods and Protocols serves as an ideal guide for researchers attempting to further our knowledge of this vibrant area of study.

Includes cutting-edge techniques Provides step-by-step detail essential for reproducible results Contains key implementation advice from the experts

Inhalt

Enabling Technologies for the Dissection of Inositol Pyrophosphate Physiology.- Cell Extraction and Enrichment of Inositol Polyphosphate and Inositol Pyrophosphates Using Titanium Dioxide.- Analysis of Inositol Phosphates via Polyacrylamide Gel Electrophoresis (PAGE).- Enzymatic Synthesis of Inositol Pyrophosphates.- Determination of Steady-State Inositol Phosphate Profile of the Liverwort, Marchantia polymorpha Using SAX-HPLC.- Analysis of Inositol Phosphates, Diphosphoinositol Phosphates, and Their Analogues by HPLC on an Acid-Compatible Stationary Phase by Complexation with Ferric Ion.- Using HILIC-MS/MS Method to Study Inositol Pyrophosphate.- Capillary Electrophoresis Mass Spectrometry for Inositol (Pyro)Phosphate Profiling.- Absolute Configurations of Inositol Poly- and Pyrophosphates Assigned by Nuclear Magnetic Resonance Spectroscopy.- (PP)-InsP Affinity Probes for Target Characterization by Immunoblotting and Mass Spectrometry.- Enrichment Workflow for Pyrophosphoproteome Analysis.- The Use of Grating-Coupled Interferometry to Study Inositol Pyrophosphate-Protein Interactions.- Inositol Phosphate Kinase Architecture: Practical Approaches and Lessons Learned.- Visualizing and Identifying Inositol Pyrophosphate Isomers In Crystallo.- An In Vivo Yeast-Based Activity Assay for the PPIP5K Family.- Hydroponic Cultivation Systems to Study Nutrient-Dependent Changes in Inositol Polyphosphate Metabolism in Plants.- A Simple RUBY Reporter System in Nicotiana benthamiana for Studying Inositol Pyrophosphate Dynamics.- Knockout Mice as an Experimental Model to Study the Biology of Inositol Pyrophosphates.- IP6 Kinase Inhibitor Screening Assays.