Methods for binding analysis of small GTP-binding proteins with their effectors

Proteins often do not function as a single biomolecular entity; instead, they frequently interact with other proteins and biomolecules forming complexes. There is increasing evidence depicting the essentiality of protein-protein interactions (PPIs) governing a wide array of cellular processes. Thus, it is crucial to understand PPIs. Commonly used approaches like genetic (e.g., Yeast Two-Hybrid, Y2H), optical (e.g., Surface Plasmon Resonance, SPR; Fluorescence Resonance Energy Transfer, FRET), and biochemical have rendered ease in developing interactive protein maps as freely available information in protein databases on the web. The underlying basis of traditional protein interaction analysis is the core of biochemical methodologies providing direct evidence of interactions. Co-Immunoprecipitation (Co-IP) is a powerful biochemical technique that facilitates identifying novel interacting partners of a protein of interest in vivo, allowing specific capture of their complexes on an immunoglobulin. Here, using Arf-like (Arl) GTPase-8b (Arl8b) and Pleckstrin Homology Domain-Containing Family MMember 1 (PLEKHM1) as an example of small GTPase-effector pair, we provide a detailed protocol for performing Y2H and Co-IP assays to confirm the interaction between a small GTPase and its effector protein.