KRAS, 1st ed. 2024
Methods and Protocols
Methods in Molecular Biology Series, Vol. 2797

This volume details protocols ranging from high yield production metabolically labeled KRAS for NMR studies to approaches that quantify engagement of novel molecules that bind KRAS in live cells. Chapters focus on protein production and characterization, biochemical assays, cell-based assays, KRAS-membrane interactions, targeting KRAS, and cell models. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, KRAS: Methods and Protocols aims to provide methods that will be instrumental in the development of future clinically approved KRAS therapeutics.

1         KRAS as a therapeutic target

           Dwight V. Nissley, Andrew G. Stephen, Ming Yi, and Frank McCormick

2         The abundance of KRAS and RAS gene mutations in cancer

           Edward C. Stites

3         Production of isotopically labeled KRAS4b

           Troy Taylor and William Gillette

4         Nucleotide Exchange on RAS Proteins using Hydrolysable and Non-Hydrolysable Nucleotides

           Timothy Waybright

5         Crystallographic studies of KRAS in Complex with Small Molecules and RAS-binding Proteins

           Albert H. Chan and Dhirendra K. Simanshu

6         Considerations around Structure-Based Drug Discovery for KRAS using DOCK

           Mayukh Chakrabarti, Y. Stanley Tan, and Trent Balius

7         Measurement of KRAS GTPase activity

           Dana Rabara and Andrew G. Stephen

8         Affinity Measurement of Non-covalent Interactions of the covalent KRAS G12C GDP inhibitor MRTX849 to RAS Isoforms using Surface Plasmon Resonance

           Patrick Alexander and Andrew G. Stephen

9         1D and 2D NMR for KRAS:ligand binding

           Gabriel Cornilescu

10       Luminescence based techniques for KRAS thermal stability monitoring

           Kari Kopra

11       MALDI-TOF mass spectrometry-based assay for measuring covalent target engagement of KRAS G12C inhibitors

           Marcin Dyba, John-Paul Denson, and Anna E. Maciag

12       KRAS4b:RAF-1 Homogenous Time-Resolved Fluorescence Resonance Energy Transfer Assay for Drug Discovery

13       Biophysical characterization of RAS-SOS Complexes by Native Mass Spectrometry

           Sangho Yun, Elena Scott, and Arthur Laganowsky

14       Profiling complex RAS-effector interactions using NMR spectroscopy

           Regina Strakhova and Matthew J. Smith

15       Probing RAS Function Using Monobody and NanoBiT Technologies

           Michael Whaby, Rakesh Sathish Nair, and John O'Bryan

16       Analysis of the guanine nucleotide-bound state of KRAS by ion-pair reversed-phase high-performance liquid chromatography

           Makoto Araki and Kenji Kontani

17       Real-time monitoring of RAS activity using in vitro and in-cell NMR spectroscopy

           Qingci Zhao, Ichio Shimada, and Noritaka Nishida

18       Studying RAS Interactions in live cells with BRET

           John Columbus and Tommy Turbyville

19       FLIM-FRET Protein-Protein Interaction Assay

           Pedro Andrade Bonilla and Rebika Shrestha

20       High-throughput cell-based screening of small molecule KRAS signaling inhibitors using a homogeneous time resolved fluorescence (HTRF) assay

           Brian P. Smith, Megan Rigby, Roger Ma, and Anna E. Maciag

21       A method to conditionally measure target engagement at intracellular RAS and RAF complexes.

           James D. Vasta, Ani Michaud, J. Aaron Crapster, and Matthew B. Robers

22       FLAG-KRAS4B as a model system for KRAS4B proteoform and PTM evaluation by mass spectrometry 

           Robert A. D’Ippolito, Grace M. Scheidemantle, Brian Smith, Katie Powell, Scott Eury, Abigail Neish, Jennifer Mehalko, Lauren Beaumont, Nicole Fer, Vanessa Wall, William Burgan, Anna E. Maciag, Dominic Esposito, and Caroline J. DeHart

23       Creation of an isogenic H/N/KRAS-less mouse embryonic fibroblast cell line panel derived from a size sorted diploid clone

           William Burgan

24       Quality Control of an Isogenic H/N/KRAS-less Mouse Embryonic Fibroblast Cell Line Panel 

           Bingfang Xu and Katie Powell

25       A Facile Method to Append a Bio-ID Tag to Endogenous Mutant Kras Alleles

           Konstantin Budagyan, Alexa C. Cannon, and Jonathan Chernoff

Includes cutting-edge methods and protocols

Provides step-by-step detail essential for reproducible results

Contains key notes and implementation advice from the experts