Improve Research Reproducibility A Bio-protocol resource
Concise Method
tooltip

In vitro kinase assay

XT Xiaolong Tang
GL Guo Li
LS Lei Shi
FS Fengting Su
MQ Minxian Qian
ZL Zuojun Liu
YM Yuan Meng
SS Shimin Sun
JL Ji Li
BL Baohua Liu
request Request a Protocol
ask Ask a question
Favorite

To prepare the AMPK kinase complex, FLAG-AMPK was transfected into HEK293 cells which were treated with AICAR (10 mM) for 12 h to stimulate AMPK activation and then immunoprecipitated (IPed) using anti-FLAG agarose beads according to the manufacturer’s instructions. The in vitro kinase assays were conducted in a kinase reaction buffer (50 mM Tris-HCl pH 7.4, 1 mM dithiothreitol (DTT), 10 mM MgCl2, 0.5 M ATP) containing the IPed AMPK complex and active GSK-3β (100 ng) plus 2 µg soluble various forms of GST-SIRT7 proteins. Reactions were incubated at 30 °C for 1 h and then terminated by the addition of sodium dodecyl sulfate (SDS) sample buffer. The phosphorylated SIRT7 was assessed by western blotting.

Copyright and License information: The Author(s) ©2021
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

post Post a Question
0 Q&A