rKRAS was exchanged in 20 mM tris (pH 7.5), 150 mM NaCl, and 5 mM β-mercaptoethanol using PD10 desalting columns (GE Healthcare). rKRAS was incubated for 1 hour at 37°C with rRRSP at a molar ratio of 1:100 (rKRAS/rRSSP). For the EDTA-mediated nucleotide exchange assay, 2 μM rKRAS and 2 μM rKRAS* were mixed with 4 μM mant-GTP (Thermo Life Technologies) or mant-GppNHp (Jena Bioscience) in assay buffer [20 mM tris (pH 7.5), 150 mM NaCl, 5 mM β-mercaptoethanol, 10 mM MgCl2, and 20 mM EDTA] and dispensed into a 384-well plate. Fluorescence was measured every 10 s for 5 min at excitation/emission set to 360 nm/440 nm at 25°C in a SpectraMax M3 plate reader (Molecular Devices). Data were fit in GraphPad Prism 6.0 (GraphPad Software Inc.) to a one-phase exponential association curve to determine the observed nucleotide exchange rate kobs. Data were reported as means ± SD from three independent biological replicates.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.