Enzchek phosphate assay kit

ATPase assays were performed using the EnzChek phosphate assay kit (Invitrogen) with motor concentrations in the 5 to 50 nM range and 25 nM to 20 µM polymerized tubulin. ATPase assays were performed in 25 mM HEPES, 50 mM potassium acetate, 5 mM magnesium acetate, 1 mM EGTA, and 1 mM DTT, pH 7.5. k_cat and K_0.5,MT were calculated from fitting the Michaelis–Menten equation to ATPase data taken at a >10-fold excess of microtubules.
Pre-steady-state kinetics of 2′ deoxy 3′ MANT-ATP (2′dmT) binding, nucleotide-dependent microtubule release and Pi release were performed on a KinTex SF-2004 stopped-flow apparatus as previously described [85 (link)].
Binding of the fluorescent nucleotide analogue 2′dmT to 4 : 1 microtubules:motor complex was measured by mixing with an excess of fluorescent nucleotide in a KinTek F-300X stopped flow at 20°C, with instrument dead time of 1.2 ms. Samples were rendered nucleotide-free prior to mixing by incubating for 20 min with 0.2 U ml⁻¹ apyrase (Type VII, Sigma Aldrich, St Louis, MO). Fluorescence enhancement of the MANT-fluorophore was monitored by energy transfer from vicinal tryptophans by exciting at 295 nm and monitoring 90^o from the incident beam through a 450 nm broad bandpass filter (Omega Optical, Brattleboro, VT). Data were subjected to linear least squares fitting.

Five grams (g) of leaf tissue from each A. hybridus population finely powdered with liquid nitrogen. EPSPS enzyme extraction was performed following the protocol described by Dayan et al. [47 (link)] The total soluble protein (TPS) in the extract (EPSPS basal activity in absence of glyphosate) was determined by the Bradford assay [48 (link)], using a Kit for Protein Determination (Sigma-Aldrich, Madrid, Spain) following manufacturer’s instructions. The specific EPSPS activity was assayed in the presence of glyphosate (0, 0.1, 1, 10 100, and 1000 µM) using the EnzChek Phosphate Assay Kit (Invitrogen, Carlsbad, CA, USA) to determine the EPSPS inhibition by 50% (I₅₀). Five replications of each population per glyphosate concentration were assayed. EPSPS inhibition was expressed as a percentage relative to the control (absence of glyphosate)

The GAP assay was carried out using the EnzChek Phosphate Assay Kit (Invitrogen), similar to previous studies [12 (link),50 (link)]. Briefly, RAB proteins were purified and incubated with 15-fold molar excess of GTP at room temperature for 30 min. After incubation, free nucleotides were removed by passing a desalting column. GTP-preloaded Rabs and purified TBC domain of TBC1D23 were prepared in two different solutions. The reaction was initiated by mixing the two solutions, and the final solution contained 20 mM HEPES (pH 7.5), 150 mM NaCl, 10 mM MgCl2, 200 mM 2-amino-6-mercapto-7-methylpurine riboside (MESG), 5 units of purine nucleoside phosphorylase, 20 mM Rab, and various amounts of TBC1D23. The absorbance at 360 nm was continuously monitored, and kinetics was determined similarly to previous studies [50 (link)].

ATPase activity was determined as described in the protocol of the EnzChek phosphate assay kit (Invitrogen). The tetramer cohesin of a final concentration of 50 nM was dissolved with 50 mM NaCl. If dsDNA is presented, 700 nM 40 bp dsDNA was used. The reaction was initialised by adding ATP to a final concentration of 1.3 mM (final reaction volume: 150ul). The OD at 360 nm was monitored every 30 s for 90 min using a PHERAstar FS. ΔΑU at 360 nm was translated to Pi release using an equation derived by a standard curve of KH₂PO₄ (EnzChek kit). Rates were calculated from the slope of the linear phase (first 10 min). At least two independent biological experiments were performed for each experiment.

KRAS was loaded with GTP nucleotide by incubating 100 µM protein with 10 mM EDTA and 500 µM ultra-pure GTP (Thermo Scientific, R0461) for 2 h at room temperature with slow shaking (150 rpm). Unbound GTP was removed using 2 ml Zeba column with 7 kDa molecular weight cutoff (Thermo Scientific, 89889) and exchanged into 30 mM Tris pH = 7.5 buffer containing 1 mM DTT. The protein sample was immediately used in GTP hydrolysis in vitro assays using EnzChek Phosphate Assay Kit (Invitrogen, E6646) following manufacturer instructions. Reactions were performed in 384-well clear microplates with 30 mM Tris pH = 7.5 buffer containing 1 mM DTT. For each sample reaction, final concentration of the following reagents was added: 200 μM MESG, 1 U/ml PNP, 50 µM KRAS^GTP. To start reaction, ADAP1 (25 µM final concentration) plus 40 mM MgCl₂ or buffer plus 40 mM MgCl₂ were added quickly and measured absorbance at 360 nm every 4 s for 1 h. Data were analyzed by subtracting the background (no-substrate control) at each measurement and graphing relative light units using GraphPad 9.0.2. Data were fit to a one-phase association curve to obtain relative GTP-hydrolysis rate (K_obs).