The nucleotide-binding assays were performed using the fluorescently labeled nucleotides mant-GTP [2′-/3′-O-(N’-methylanthraniloyl) guanosine-5′-O- triphosphate] (JenaBioscience)(17 (link)). Reactions of 100 μl were performed in 96 well-plates, with 1 μM of recombinant protein incubated with 0.5 μM of mant-nucleotides in buffer containing 20 mM HEPES pH 8.0, 200 mM KCl, 5 mM MgCl2, 1 mM DTT for 10 min at 30°C. The reaction mixture was then excited at 355 nm with a xenon lamp, and emission spectra were recorded between 385 and 600 nm with a 5-nm increment step using a Synergy 4 spectrophotometer (BioTek). All data were processed with Microsoft Office Excel and GraphPad PRISM.
Mant gtp
Manufactured by Jena Biosciences
Sourced in Germany
Mant-GTP is a fluorescent GTP analog synthesized by Jena Biosciences. It is used as a tool compound for research purposes.
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Lab products found in correlation
2 protocols using mant gtp
1
Fluorescent Nucleotide-Binding Assay
2
Evaluating GGDEF Protein Functionality
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To verify the functionality of mutated SoGGDEF proteins they were tested for GTP binding using MANT-GTP (Jena Bioscience, Germany). Proteins were diluted to 1 µM and 100 µl of each sample was added in triplicates into a white round bottom 96-well plate before MANT-GTP with a final concentration of 1 µM was added. The plate was then incubated for 30 min at room temperature on a plate shaker before the fluorescence (excitation: 295 nm, emission: 448 nm) was measured in a Tecan Infinite M200 plate reader (Tecan, Switzerland).
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