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This volume compiles new experimental approaches and concepts focusing mostly, but not solely, on ways to manipulate and regulate Ras activity and its downstream signaling output. Chapters detail standard methodologies, biochemical methods, Ras processing trafficking and localization, Ras signaling and inhibition, and in vivo models for studying Ras function. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, application details for both the expert and non-expert reader, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and accessible, Ras Activity and Signaling: Methods and Protocols aims to provide support and guidance to lab workers in their work on Ras GTPases and in the design of new projects requiring novel methodologies.
List of contents
Ras Variant Biology and Contributions to Human Disease.- Regulation of the Small GTPase Ras and its Relevance to Human Disease.- Precise Characterization of KRAS4B Proteoforms by Combining Immunoprecipitation with Top-Down Mass Spectrometry.- Absolute Quantitation of GTPase Protein Abundance.- Validation of Isoform- and Mutation-specific RAS Antibodies.- Production and Membrane Binding of N-terminally Acetylated, C-terminally Farnesylated and Carboxymethylated KRAS4b.- Active GTPase Pulldown Protocol.- Methods to Monitor Ras Activation State.- NMR Detection Methods for Profiling RAS Nucleotide Cycling.- Ras Diffusion and Interactions with the Plasma Membrane Measured by FRAP Variations.- Understanding Ras Spatial Cycles through Reaction-Diffusion Simulations.- Super-Resolution Imaging and Spatial Analysis of RAS on Intact Plasma Membrane Sheets.- FLIM-FRET Analysis of Ras Nanoclustering and Membrane-Anchorage.- Assessment of Plasma Membrane Fatty Acid Composition and Fluidity Using Imaging Flow Cytometry.- Spatiotemporal Imaging of Small GTPase Activity using Conformational Sensors for GTPase Activity (COSGA).- Using BioID to Characterize the RAS Interactome.- Probing RAS function with Monobodies.- Detection of Endogenous RASSF1A Interacting Proteins.- Mathematical Modeling to Study KRAS Mutant-Specific Responses to Pathway Inhibition.- A Facile Method to Engineer Mutant Kras Alleles in an Isogenic Cell Background.- RASless MEFs as a Tool to Study RAS-dependent and -independent Functions.- Generation of Patient-derived Colorectal Cancer Organoids for RAS Studies.- Ras-GEF Mouse Models for the Analysis of Ras Biology and Signaling .- Studying Metabolic Abnormalities in the Costello Syndrome HRAS G12V Mouse Model: Isolation of Mouse Embryonic Fibroblasts and Their In Vitro Adipocyte Differentiation.- Dissecting Oncogenic RAS Signalling in Melanoma Development in Genetically Engineered Zebrafish Models.- Ras, Ral, and Rap1 in C. elegans.
