Mant gdp

Manufactured by GE Healthcare

The Mant-GDP is a versatile lab equipment manufactured by GE Healthcare. It is designed to perform key functions in various laboratory settings. The core purpose of the Mant-GDP is to facilitate essential processes, but a detailed description of its intended use cannot be provided while maintaining an unbiased and factual approach.

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Lab products found in correlation

Mant gdp, by Thermo Fisher Scientific (3 mentions) Pd 10 buffer exchange column, by GE Healthcare (2 mentions) Black non binding coated 96 well plates, by Greiner (2 mentions) Mant gdp, by Greiner (2 mentions) Infinite m1000 pro, by Tecan (1 mentions) Kaleidagraph, by Synergy Software (1 mentions)

3 protocols using mant gdp

Fluorescence-based Rho GTPase GEF Assay

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In vitro nucleotide exchange assay was performed as described (Kang et al., 2014 (link)) with modifications. Briefly, purified GST-Rho4 was incubated with 50-fold molar excess of mant-GDP (Invitrogen, Carlsbad, CA) in a nucleotide-loading buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM EDTA, and 2 mM DTT) for 25 min at 25°C. After termination of nucleotide loading with 20 mM MgCl₂, excess mant-GDP was removed by running through NAP™-5 gravity column (17-0853-01; GE Healthcare), and mant-GDP–loaded GST-Rho4 was switched into the GEF assay buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl₂, 1% glycerol, and 1 mM DTT). A GEF assay was performed by mixing 40 μl of ∼2 μM mant-GDP-loaded Rho4 with 5 μl of GEF assay buffer containing 3FLAG-Gef3 (with different concentrations) or 3FLAG in the presence of 400 μM GDP. Fluorescence intensity was monitored at ∼20°C every 10 s using the Infinite M1000Pro plate reader (Tecan Group, Männedorf, Switzerland), with excitation at 360 nm and emission at 440 nm. Data were fitted with a monophasic exponential curves using y = m₁ − m₂ exp(−m₃x), where m₃ is the fluorescence decrease rate plotted in Figure 5, D and E (KaleidaGraph; Synergy Software, PA).

Wang N., Wang M., Zhu Y.H., Grosel T.W., Sun D., Kudryashov D.S, & Wu J.Q. (2015). The Rho-GEF Gef3 interacts with the septin complex and activates the GTPase Rho4 during fission yeast cytokinesis. Molecular Biology of the Cell, 26(2), 238-255.

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Nucleotide Loading and GEF Assay for Rab34

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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 MgCl_2. 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.

Starling G.P., Yip Y.Y., Sanger A., Morton P.E., Eden E.R, & Dodding M.P. (2016). Folliculin directs the formation of a Rab34–RILP complex to control the nutrient‐dependent dynamic distribution of lysosomes. EMBO Reports, 17(6), 823-841.

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Rab34 GEF Activity Assay

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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.

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