Titlebook: Ras Activity and Signaling; Methods and Protocol Ignacio Rubio,Ian Prior Book 2021 Springer Science+Business Media, LLC, part of Springer N

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Super-Resolution Imaging and Spatial Analysis of RAS on Intact Plasma Membrane Sheetscluster size, clustered fraction, and population distribution can be obtained by the univariate spatial distribution analysis. Intermolecular associations between different RAS isoforms, RAS and various PM lipids, as well as RAS and diverse effectors can be quantified via bivariate co-localization analysis.

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Book 2021s 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, chapte

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Validation of Isoform- and Mutation-Specific RAS Antibodiesd for well-validated antibodies to evaluate the function and expression of the different RAS isoforms. Here we describe our experimental approaches to characterize RAS antibodies for their isoform- and mutant-specificity for use in immunoblot analyses.

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Production and Membrane Binding of N-Terminally Acetylated, C-Terminally Farnesylated and Carboxymetassociate with the plasma membrane and fulfill its function in cell signaling. We describe here the production of recombinant KRAS4b from our modified baculovirus/insect cell expression system that accurately incorporates these in vivo modifications to allow experiments that anchor KRAS4b to membrane mimetics (e.g., nanodiscs and liposomes).

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Assessment of Plasma Membrane Fatty Acid Composition and Fluidity Using Imaging Flow Cytometrythis chapter, we describe in vitro methods to modulate membrane phospholipid fatty acid composition of cultured cells using fatty acids complexed to bovine serum albumin (BSA). Furthermore, we describe a method to quantify the biophysical properties of plasma membranes in live cells using Di-4-ANEPPDHQ (Di4) and image-based flow cytometry.

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Spatiotemporal Imaging of Small GTPase Activity Using Conformational Sensors for GTPase Activity (CO for imaging small GTPase activity in the cell. Conformational changes upon GDP/GTP binding can be visualized directly in solution, on beads, or in live cells using COSGA by fluorescence lifetime imaging microscopy (FLIM) technique. Herein, we describe the construction of COSGA for imaging K-Ras GTPase activity in live cells.

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Precise Characterization of KRAS4B Proteoforms by Combining Immunoprecipitation with Top-Down Mass Serization of intact KRAS4B proteoforms. We provide detailed protocols for the IP, LC-MS/MS, and data analysis comprising a successful IP-TDMS assay in the contexts of cancer cell lines and tissue samples.

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NMR Detection Methods for Profiling RAS Nucleotide Cyclingmultiplex these assays, allowing for the precise monitoring of activation states for mixtures of RAS oncoproteins or other RAS superfamily GTPases. Here, we describe the protocols necessary to express and purify isotopically labeled RAS and detail how to carry out an RT-NMR assay on a singular RAS protein or on a mixture of small GTPases.

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Ras Diffusion and Interactions with the Plasma Membrane Measured by FRAP Variations the dynamics of the association of N-Ras with the plasma membrane of living cells and their dependence on several parameters (cholesterol, clustering of raft proteins, and palmitoylation/depalmitoylation).