GTPase activity of dynamin 2 was determined by monitoring release of free orthophosphate using malachite green assay (47 (link)). The malachite green reagent was prepared by mixing solution A (17 mg of Malachite Green Carbinol base dye (229105, Merck) in 20 ml 1 N HCl) and Solution B (0.5 g Ammonium molybdate (277908, Merck) in 7 ml 4 N HCl) with filling up to 50 ml by MilliQ water followed by filtration through 0.45 μm membrane (S-2504, KURABO). In the assay, 0.2 μM dynamin in the presence of BIN1 at different molar ratio was mixed with 1 mM GTP in GTPase reaction buffer (10 mM Hepes, 2 mM MgCl2, 50 mM NaCl, pH 7.5) with or without 0.005 μg/μl lipid nanotubes and incubated for 5 min at 37 °C. After the reaction was stopped on ice for 10 min, 160 μl of malachite green reagent was added to the 40 μl of the reaction mix in 96-well plate (442404, Thermo Fisher Scientific). After 5 min shaking at 1200 rpm with Digital MicroPlate Genie Pulse (Scientific Industries, Inc), released orthophosphate was colorimetrically quantified by measuring OD 650 nm using a microplate reader (SH-1000, CORONA ELECTRIC).
S 2504
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The S-2504 is a laboratory equipment designed for scientific analysis and research. It is a multi-purpose device that can be used in various applications. The core function of the S-2504 is to perform precise measurements and data collection.
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2 protocols using s 2504
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Dynamin-2 GTPase Activity Assay
2
Dynamin 2 GTPase Activity Assay
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GTPase activity of dynamin 2 was determined by monitoring release of free orthophosphate using malachite green assay as described previously (Fujise et al., 2020) (link).
The malachite green reagent was prepared by mixing solution A (17 mg of Malachite Green Carbinol base dye (229105, Merck) in 20 mL 1 N HCl) and Solution B (0.5 g Pathogenic evaluation of congenital myopathy MilliQ water followed by filtration through 0.45 ߤm membrane (S-2504, KURABO). In the assay, 0.2 ߤM dynamin in the presence of BIN1 at different molar ratio was mixed with 1 mM GTP in GTPase reaction buffer (10 mM Hepes, 2 mM MgCl 2 , 50 mM NaCl, pH 7.5) with or without 0.005 ߤg/ߤL lipid nanotubes and incubated for 5 min at 37 .
After the reaction was stopped on ice for 10 min, 160 ߤL of malachite green reagent was added to the 40 ߤL of the reaction mix in 96 well plate (442404, Thermo Fisher Scientific). After 5 min shaking at 1200 rpm with Digital MicroPlate Genie Pulse (Scientific Industries, Inc.), released orthophosphate was colorimetrically quantified by measuring OD 650 nm using a microplate reader (SH-1000, CORONA ELECTRIC).
The malachite green reagent was prepared by mixing solution A (17 mg of Malachite Green Carbinol base dye (229105, Merck) in 20 mL 1 N HCl) and Solution B (0.5 g Pathogenic evaluation of congenital myopathy MilliQ water followed by filtration through 0.45 ߤm membrane (S-2504, KURABO). In the assay, 0.2 ߤM dynamin in the presence of BIN1 at different molar ratio was mixed with 1 mM GTP in GTPase reaction buffer (10 mM Hepes, 2 mM MgCl 2 , 50 mM NaCl, pH 7.5) with or without 0.005 ߤg/ߤL lipid nanotubes and incubated for 5 min at 37 .
After the reaction was stopped on ice for 10 min, 160 ߤL of malachite green reagent was added to the 40 ߤL of the reaction mix in 96 well plate (442404, Thermo Fisher Scientific). After 5 min shaking at 1200 rpm with Digital MicroPlate Genie Pulse (Scientific Industries, Inc.), released orthophosphate was colorimetrically quantified by measuring OD 650 nm using a microplate reader (SH-1000, CORONA ELECTRIC).
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