Small GTPases act as molecular switches regulating various cellular processes by cycling between the GDP- and GTP-bound states. Several methods, including radioisotope-based nucleotide exchange assays, effector-binding pull-down assays, and fluorescence-based biosensor methods, have been developed to assess the activation of small GTPases. In vitro techniques mainly provide quantitative insights, whereas live-cell imaging approaches facilitate the real-time monitoring of the activation dynamics of small GTPases. Recent advances, such as the development of fluorescence resonance energy transfer-based probes and membrane-localization sensors, have improved the spatial and temporal resolution of small GTPase activation dynamics. Specifically, the small GTPase activity analyzing system using a split fluorescent protein to detect membrane recruitment upon activation provides a novel approach to study small GTPases in living cells. This review comprehensively discusses various conventional and emerging small GTPase activation analysis techniques, highlighting their advantages and disadvantages in studying small GTPase activation dynamics under different cellular conditions.
