Nucleotide Exchange on RAS Proteins Using Hydrolysable and Non-hydrolysable Nucleotides

Timothy Waybright; Andrew G Stephen

doi:10.1007/978-1-0716-3822-4_4

Nucleotide Exchange on RAS Proteins Using Hydrolysable and Non-hydrolysable Nucleotides

Methods Mol Biol. 2024:2797:35-46. doi: 10.1007/978-1-0716-3822-4_4.

Authors

Timothy Waybright¹, Andrew G Stephen²

Affiliations

¹ NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, MD, USA. Timothy.Waybright@nih.gov.
² NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, MD, USA.

PMID: 38570451
DOI: 10.1007/978-1-0716-3822-4_4

Abstract

Biochemical and biophysical assays using recombinant RAS require the protein to be in either the active or inactive state. Here we describe methods to exchange the nucleotide present in the purified RAS protein with either GDPβS, GppNHp, or GTP depending on the assay requirement. In addition, we also describe the HPLC method used to validate the exchange process and provide information on the efficiency of the nucleotide exchange.

Keywords: Chromatography; Ion pairing; Nucleotide exchange; RAS.

MeSH terms

Guanosine Diphosphate
Guanosine Triphosphate / metabolism
ras Proteins* / genetics
ras Proteins* / metabolism

Substances

Guanosine Triphosphate
ras Proteins
Guanosine Diphosphate