Ras Activity and Signaling, 1st ed. 2021 Methods and Protocols Methods in Molecular Biology Series, Vol. 2262

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.

Part I: Generic Ras Biology

1. Ras Variant Biology and Contributions to Human Disease

Ian A. Prior

2. Regulation of the Small GTPase Ras and its Relevance to Human Disease

Part II: Methods in Ras Biochemistry

3. Precise Characterization of KRAS4B Proteoforms by Combining Immunoprecipitation with Top-Down Mass Spectrometry

Lauren M. Adams, Caroline J. DeHart, and Neil L. Kelleher

4. Absolute Quantitation of GTPase Protein Abundance

Fiona E. Hood, Yasmina M. Sahraoui, Rosalind E. Jenkins, and Ian A. Prior

5. Validation of Isoform- and Mutation-specific RAS Antibodies

Andrew M. Waters and Channing J. Der

6. Production and Membrane Binding of N-terminally Acetylated, C-terminally Farnesylated and Carboxymethylated KRAS4b

Simon Messing, Constance Agamasu, Matt Drew, Caroline DeHart, Andrew G. Stephen,  and William K. Gillette

7. Active GTPase Pulldown Protocol

Martin J. Bakerand Ignacio Rubio

8. Methods to Monitor Ras Activation State

Kari Kopra and Harri Härmä

9. NMR Detection Methods for Profiling RAS Nucleotide Cycling

Ryan C. Killoran and Matthew J. Smith

Part III: Methods in Ras Processing, Trafficking, and Localization

Orit Gutman, Marcelo Ehrlich, and Yoav I. Henis

11. Understanding Ras Spatial Cycles through Reaction-Diffusion Simulations

Malte Schmickand Philippe I. H. Bastiaens

12. Super-Resolution Imaging and Spatial Analysis of RAS on Intact Plasma Membrane Sheets

Yong Zhou and John F. Hancock

13. FLIM-FRET Analysis of Ras Nanoclustering and Membrane-Anchorage

Hanna Parkkola, Farid Ahmad Siddiqui, Christina Oetken-Lindholm, and Daniel Abankwa

14. Assessment of Plasma Membrane Fatty Acid Composition and Fluidity Using Imaging Flow Cytometry

Natividad R. Fuentes, Michael L. Salinas, Xiaoli Wang, Yang-Yi Fan, and Robert S. Chapkin

15. Spatiotemporal Imaging of Small GTPase Activity using Conformational Sensors for GTPase Activity (COSGA)

Yao-Wen Wu

Part IV: Methods in Ras Signaling and Inhibition

16. Using BioID to Characterize the RAS Interactome

Hema Adhikari and Christopher M. Counter

17. Probing RAS function with Monobodies

Imran Khan and John P. O’Bryan

18. Detection of Endogenous RASSF1A Interacting Proteins

Howard Donninger, Desmond Harrell-Stewart, and Geoffrey J. Clark

19. Mathematical Modeling to Study KRAS Mutant-Specific Responses to Pathway Inhibition

Edward C. Stites

20. A Facile Method to Engineer Mutant Kras Alleles in an Isogenic Cell Background

Konstantin Budagyanand Jonathan Chernoff

Carmen G. Lechuga, Marina Salmón, Guillem Paniagua, Carmen Guerra, Mariano Barbacid, Matthias Drosten

Part V: Methods in vivo Ras Biology

22. Generation of Patient-derived Colorectal Cancer Organoids for RAS Studies

Dirk Schumacher, Joseph L. Regan, Dorothea Przybilla, and Reinhold Schäfer

23. Ras-GEF Mouse Models for the Analysis of Ras Biology and Signaling

24. Studying Metabolic Abnormalities in the Costello Syndrome HRAS G12V Mouse Model: Isolation of Mouse Embryonic Fibroblasts and Their In Vitro Adipocyte Differentiation

Miray Fidan, Saravanakkumar Chennappan, and Ion Cristian Cirstea

25. Dissecting Oncogenic RAS Signalling in Melanoma Development in Genetically Engineered Zebrafish Models

Andrew P. Badrock and Adam Hurlstone

26. Ras, Ral, and Rap1 in C. elegans

Neal R. Rasmussen and David J. Reiner

Includes cutting-edge methods and protocols

Provides step-by-step detail essential for reproducible results

Contains key notes and implementation advice from the experts