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Updated: Jun 14, 2025
Assaying Protein Kinase Activity with Radiolabeled ATP
Published on: May 26, 2017
Protocol for detecting SnRK2 kinase activity in plants by immunoblotting, in-gel assay, band shift, and
Qingzhong Li1, Xianping Yuan2, Yang Zhao2
1Key Laboratory of Plant Carbon Capture, Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
Abstract:
The SNF1-regulated protein kinase 2s (SnRK2s) are activated by phytohormone abscisic acid (ABA) and osmotic stress to control plant growth and stress responses; however, assessing SnRK2 activity is challenging. Here, we present a protocol to detect SnRK2 activity in plants. We describe steps for performing immunoblotting with anti-phospho-S175-SnRK2 antibody, in-gel kinase assay, band-shift assay, and immunoprecipitated kinase assay. Immunoblotting and in-gel kinase assays are suitable for evaluating endogenous SnRK2 activity, whereas band-shift and immunoprecipitated kinase assays are applicable to assess tagged SnRK2 activity in transgenic lines. For complete details on the use and execution of this protocol, please refer to Li et al.,1 Li et al.,2 and Yuan et al.3.
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