Mant gtp

Manufactured by Thermo Fisher Scientific

Sourced in France, United States

MANT-GTP is a fluorescent analogue of the guanosine triphosphate (GTP) molecule. It is used as a tool in biochemical research to study the activity and regulation of GTP-binding proteins.

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Lab products found in correlation

Glomax luminometer, by Promega (1 mentions) Pgex 6p 1, by GE Healthcare (1 mentions) Pmal c2, by New England Biolabs (1 mentions) Flx800 fluorescence microplate reader, by Agilent Technologies (1 mentions) Synergy 2, by Agilent Technologies (1 mentions) Mant atp, by Thermo Fisher Scientific (1 mentions) Mant gdp, by Thermo Fisher Scientific (1 mentions) Synergy ht plate reader, by Agilent Technologies (1 mentions) Varioskan flash, by Thermo Fisher Scientific (1 mentions)

7 protocols using mant gtp

GAP Domain Protein Binding Assay

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The Spin6 GAP domain fragment was cloned into the GST fusion vector pGEX-6p-1 (GE Healthcare). OsRac1 cDNA was cloned into the MBP vector pMAL-c2 (New England BioLabs). The proteins were expressed and purified according to the manufacturer’s protocols for GAP activity assay. OsRac1 protein at 1 μM was loaded with 10 μM mant-GTP (Molecular Probes, M12415) in the following buffer: 20 mM Tris/HCl pH 8.0, 50 mM NaCl, and 1 mM EDTA. After 10 min at 25°C, the mant-GTP loaded OsRac1 was used for the GAP activity assay by adding 5 μM SPIN6GAP or GST (control) proteins in reaction buffer (200 mM Tris-Cl pH 8.0, 500 mM NaCl, 10 mM EDTA). Samples were immediately placed into a Glo-MAX luminometer (Promega) for fluorescence detection (at 10 min intervals for 30 min) at 25°C. Each reaction was repeated three times, and means and standard errors are presented.

Liu J., Park C.H., He F., Nagano M., Wang M., Bellizzi M., Zhang K., Zeng X., Liu W., Ning Y., Kawano Y, & Wang G.L. (2015). The RhoGAP SPIN6 Associates with SPL11 and OsRac1 and Negatively Regulates Programmed Cell Death and Innate Immunity in Rice. PLoS Pathogens, 11(2), e1004629.

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Fluorescence-based guanine-nucleotide exchange assay

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Fluorescence-based in vitro guanine-nucleotide exchange assays were performed using Mant-GTP (Molecular Probes, Life Technologies, St-Aubin, France) in an FLX 800 microplate fluorescence reader (BioTek Instruments, Colmar, France) at 25°C, as described (Bouquier et al., 2009 (link)). Nucleotide exchange was determined by measuring Mant-GTP loading on GDP-preloaded Rac1, Cdc42, or RhoA GTPases using 1 μM GEF (0.5 μM for Dbl). The relative Mant fluorescence (λ_ex = 360 nm and λ_em = 460 nm) was monitored for 30 min, and measurements were taken every 15 s.

Jaudon F., Raynaud F., Wehrlé R., Bellanger J.M., Doulazmi M., Vodjdani G., Gasman S., Fagni L., Dusart I., Debant A, & Schmidt S. (2015). The RhoGEF DOCK10 is essential for dendritic spine morphogenesis. Molecular Biology of the Cell, 26(11), 2112-2127.

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MANT-GTPγS Binding Assay for Ras Proteins

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MANT-GTP was purchased from Thermofisher. Binding of 0.5 μM MANT-GTPγS to 1–2 μM purified protein was measured at room temperature in buffer containing 20 mM Tris pH 7.5, 50 mM NaCl, plus or minus 1-10 mM MgCl₂ or 2-20 mM EDTA as indicated, in 100 μl reaction volumes in a black-bottomed microplate. Fluorescence data were collected on a BioTek Synergy 2 multi-mode reader with excitation/emission filters of 360/40 and 450/50, respectively. A time course of binding was carried out with fluorescence measurements taken every 30 s for 30 min, with protein added to nucleotide after 3 baseline measurements (which were averaged as signal at time zero). In each experiment, the fluorescence signal was normalized to time zero. Purified H-Ras G12V was used as a positive control.

Stiegler A.L, & Boggon T.J. (2018). The N-terminal GTPase domain of p190RhoGAP proteins is a pseudoGTPase. Structure (London, England : 1993), 26(11), 1451-1461.e4.

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Nucleotide Binding Assays for AtYchF1

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Nucleotide binding assays were performed as described (Rohn et al., 1999 (link)) using MBP-AtYchF1. Thirty-one mircomolar 2'/3′-O-(N-Methyl-anthraniloyl) (Mant)-GTP (Cat# M12415, Thermo Fisher Scientific, Waltham, MA, United States) or Mant-ATP (Cat# M12417, Thermo Fisher Scientific, Waltham, MA, United States) was mixed with about 100 μM MBP-AtYchF1 or MBP-only in each 160-μL reaction. For the competition assays between ppGpp and GTP/ATP for AtYchF1 binding, the Mant-GTP/-ATP:ppGpp molar ratios of 1:1, 1:2 and 1:3 were tested.

Cheung M.Y., Li X., Ku Y.S., Chen Z, & Lam H.M. (2022). Co-crystalization reveals the interaction between AtYchF1 and ppGpp. Frontiers in Molecular Biosciences, 9, 1061350.

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Fluorescence Analysis of Obg GTPase

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N-methyl-3’-O-anthranoyl-(mant)-guanine nucleotide analogs of GTP and GDP (mant-GTP and mant-GDP, respectively) were purchased from Thermo Fisher Scientific (Waltham, MA). Protein (Obg_GC or *Obg_GC, 2 μM) was incubated with either mant-GTP or mant-GDP nucleotide (0.3 μM) in buffer containing 50 mM Tris, 2 mM DTT, 1 mM EDTA, 50 mM KCl, 10% (wt/vol) glycerol, and for reactions with mant-GTP, 5 mM MgCl₂ was included [11 (link)]. All fluorescence measurements were performed at 37°C using a Synergy HT plate reader (BioTek, Shoreline, WA). These studies were carried out at least eight times over multiple days. The means for the biological replicates with corresponding standard error of the mean (SEM) are reported.

Bonventre J.A., Zielke R.A., Korotkov K.V, & Sikora A.E. (2016). Targeting an Essential GTPase Obg for the Development of Broad-Spectrum Antibiotics. PLoS ONE, 11(2), e0148222.

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MANT-GTP Binding Kinetics of AMPH-1

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(2’-(or-3’)-O-(N-methylanthraniloyl) guanosine 5’-triphosphate (MANT-GTP) was obtained from Thermo-Fisher as a frozen 5 mM stock solution. Immediately prior to use, samples of AMPH-1 were loaded on a Sephadex S200 gel filtration column equilibrated in sample buffer (25 mM Tris, pH 7.5, 100 mM KCl, 5 mM MgCl₂). The AMPH-1 dimer peak was then collcted and concentrated. Both AMPH-1 and MANT-GTP were separately diluted into sample buffer at twice the final target concentration, mixed 1:1 and then incubated for 5 min at 23 °C prior to measurement. In all cases, the final MANT-GTP concentration was 2 μM and the final AMPH-1 concentration was varied from 3–100 μM (dimer). For competition experiments, the final GTP concentration was 250 μM. Emission spectra were acquired using a photon-counting, T-format steady state fluorometer (PTI) using excitation at 356 nm with a ± 2 nm bandpass. Emission was collected from 380–500 nm using a ± 2 nm bandpass. All measurements were taken at 23 °C using a thermally jacketed cuvette holder connected to a high-precision water bath.

Kustigian L., Gong X., Gai W., Thongchol J., Zhang J., Puchalla J., Carr C.M, & Rye H.S. (2022). GTP-stimulated membrane fission by the N-BAR protein AMPH-1. Traffic (Copenhagen, Denmark), 24(1), 34-47.

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GmROP9a Nucleotide Exchange Assay

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This assay was based on previous reports. 48, 103, 104 Briefly, 1 mM GmROP9a was preloaded with unlabeled-GDP. To start the nucleotide exchange reaction, fluorescently labeled N-methylanthraniloyl (mant)-GTP (mant-GTP; M12415, Thermo Scientific) was added to the reaction buffer and the fluorescent values were recorded for 300 s. To investigate the effect of GmGEF2a on the guanine nucleotide exchange rate of GmROP9a, the different concentrations of GmGEF2a proteins (0.2 mM, 1 mM, 4 mM) was added to the reaction buffer containing the 1 mM GmROP9a-GDP and 200 nM mant-GTP. After equilibration, GDP/GTP nucleotide exchange was monitored as the increase in the relative fluorescence of fluorescent GTP upon binding to GmROP9a GTPase, as determinded using a microplate reader (Varioskan Flash, Thermo Scientific, Waltham, MA, USA).

Gao J.P., Xu P., Wang M., Zhang X., Yang J., Zhou Y., Murray J.D., Song C.P, & Wang E. (2021). Nod factor receptor complex phosphorylates GmGEF2 to stimulate ROP signaling during nodulation. Current biology : CB, 31(16).

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