Analysis of the Guanine Nucleotide-Bound State of KRAS by Ion-Pair Reversed-Phase High-Performance Liquid Chromatography

Makoto Araki; Kenji Kontani

doi:10.1007/978-1-0716-3822-4_16

Analysis of the Guanine Nucleotide-Bound State of KRAS by Ion-Pair Reversed-Phase High-Performance Liquid Chromatography

Methods Mol Biol. 2024:2797:227-236. doi: 10.1007/978-1-0716-3822-4_16.

Authors

Makoto Araki¹, Kenji Kontani²

Affiliations

¹ Department of Biochemistry, Meiji Pharmaceutical University, Tokyo, Japan.
² Department of Biochemistry, Meiji Pharmaceutical University, Tokyo, Japan. kontani@my-pharm.ac.jp.

PMID: 38570463
DOI: 10.1007/978-1-0716-3822-4_16

Abstract

Guanine nucleotides can be quantitatively analyzed by high-performance liquid chromatography (HPLC). Here we describe an ion-pair reversed-phase HPLC (IP-RP-HPLC)-based method, which enables analyzing GDP and GTP bound to small GTPases immunoprecipitated from cells. The activation status of FLAG-KRAS expressed in HEK293T cells can be investigated with the IP-RP-HPLC method. This method also can be adapted to determine the effects of compounds such as the KRAS/G12C inhibitor sotorasib on the activation status of FLAG-KRAS in the cells.

Keywords: Guanine nucleotides; High-performance liquid chromatography; Immunoprecipitation; Ion-pair reagents; KRAS.

MeSH terms

Chromatography, High Pressure Liquid / methods
Guanine Nucleotides*
HEK293 Cells
Humans
Monomeric GTP-Binding Proteins*
Proto-Oncogene Proteins p21(ras) / genetics

Substances

Guanine Nucleotides
Proto-Oncogene Proteins p21(ras)
Monomeric GTP-Binding Proteins
KRAS protein, human