The in vitro kinase assay followed the previous published method.40 (link) 100 ng purified recombinant CTR1-KD or 1 μg MPK4 was mixed with 2 μg Myelin Basic Protein (MBP, a common kinase substrate, Sigma Cat. M1891) and 1 mM ATPγS in kinase reaction buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 10 mM MgCl2, 1xRoche Complete Protease Inhibitor mixture) and different concentration of MBZ at room temperature and vortexed for 1 h. The reactions were terminated by 20 mM EDTA, and the proteins were alkylated by 1.5 μL of 50 mM PNBM (CAYMAN CHEMICAL CO, Cat. 21456–1) at room temperature and vortexed for 2 h. This step was terminated by denaturing in NuPAGE LDS Sample Buffer and 1x NuPAGE Sample Reducing Agent (Invitrogen). Samples were subjected to SDS-PAGE. The phosphorylation status of MBP, CTR1-KD and MPK4 were analyzed by western blot using Recombinant Anti-Thiophosphate ester antibody (Abcam, Cat. Ab92570, 1:5000).
Recombinant anti thiophosphate ester antibody
Manufactured by Abcam
Recombinant Anti-Thiophosphate ester antibody is a laboratory reagent used to detect and quantify the presence of thiophosphate esters in various samples. It is produced through recombinant DNA technology.
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Lab products found in correlation
2 protocols using recombinant anti thiophosphate ester antibody
1
Kinase Activity Assay with MBP Substrate
2
In Vitro Kinase Assay for CTR1-KD and MPK4
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The in vitro kinase assay followed the previous published method 49 . 100 ng purified recombinant CTR1-KD or 1 µg MPK4 was mixed with 2 µg Myelin Basic Protein (MBP, a common kinase substrate, Sigma Cat. M1891) and 1 mM ATPyS in kinase reaction buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 10 mM MgCl2, 1xRoche Complete Protease Inhibitor mixture) and different concentration of MBZ at room temperature and vortexed for 1 hour. The reactions were terminated by 20 mM EDTA, and the proteins were alkylated by 1.5 uL of 50 mM PNBM (CAYMAN CHEMICAL CO, Cat. 21456-1) at room temperature and vortexed for 2 hours. The reactions were terminated by denaturing in NuPAGE™ LDS Sample Buffer and 1x NuPAGE™ Sample Reducing Agent (Invitrogen™). Samples were subjected to SDS-PAGE. The phosphorylation status of MBP, CTR1-KD and MPK4 were analyzed by western blot using Recombinant Anti-Thiophosphate ester antibody (Abcam, Cat. Ab92570, 1:5000) .
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