Optimized conditions for GTP loading of Ras.

ABSTRACT: Ras and the small GTPase group are essential for myriad cellular processes and cycle between GDP- and GTP-loaded states to allow stringent control of downstream signaling pathways. Biochemical studies of small GTPases can therefore require a specific nucleotide-bound state. Small GTPases possess basal intrinsic activity to process GTP into GDP plus inorganic phosphate; therefore, in vitro exchange of GDP for GTP is necessary for assays on GTP-loaded states. Here, we assess the methodology of in vitro nucleotide exchange for soluble H-Ras. We begin by describing a protocol to quantify the nucleotide-bound content of H-Ras using anion exchange chromatography and use this protocol to investigate optimal strategies for loading Ras with GTP by assessing the effects of time, temperature, H-Ras c

SUBMITTER: Vish KJ 

PROVIDER: S-EPMC12719659 | biostudies-literature | 2025 Nov

REPOSITORIES: biostudies-literature