Pd 10 buffer exchange column

Thermodynamic binding parameters for the binding of nucleotides to the Src kinase domain were obtained through isothermal titration calorimetry as published^{63 (link)}. All experiments were run in a VP-ITC instrument (Micro-cal) at 25 °C. Proteins were exchanged into 20 mM Tris (pH 8.0), 250 mM NaCl, 10 mM MgCl₂ on PD-10 buffer exchange columns (GE Life-science), and diluted to 50–100 µM. Nucleotide stocks (Sigma) were made in buffer to final concentrations between 1–3 mM. The heat of binding was measured over the injection of 295 µL of drug in 10-µL steps spaced 300 sec apart. Data were fit to a one binding site model using the Origin software package (Microcal). Error bars represent the standard error of the mean of 3 independent runs. Representative enthalpograms are shown in Supplementary Figure 7.

Rab34 proteins were dialysed overnight into loading buffer consisting of 25 mM HEPES pH 7.5, 150 mM NaCl and 5 mM EDTA. Nucleotide was loaded (mant-GDP (Thermoscientific), GDP or GTPυS) by addition of a 25-fold molar excess of the nucleotide for 2 h at room temperature followed by addition of 10 mM MgCl₂. Non-bound mant-GDP was removed using a PD-10 buffer exchange column (GE Life Sciences) equilibrated in exchange buffer HEPES pH 7.5, 150 mM NaCl, 2 mM MgCl₂. For GEF assays, 300 nM Rab34 (in a volume of 100 μl) loaded with mant-GDP was incubated with various combinations and concentrations of His-FLCN-DENN and GST-RILP. Reactions were performed in black non-binding coated 96-well plates (Greiner), and mant-GDP fluorescence was monitored using 355 nm excitation and 460 nm emission ± 10 nM band pass filters on a BMG Polarstar Omega plate reader. GTP was added at a concentration of 0.3 mM or EDTA at a concentration of 10 mM at the 2-min time point. Data were acquired at 10-s intervals for 25 min. Curves are mean of duplicate samples and are smoothed using a 6-point rolling average and polynomial fitting to reduce small fluctuations in signal due to instrument noise.