{"title":"Protocol for detecting SnRK2 kinase activity in plants by immunoblotting, in-gel assay, band shift, and immunoprecipitation.","authors":"Qingzhong Li, Xianping Yuan, Yang Zhao","doi":"10.1016/j.xpro.2025.103842","DOIUrl":null,"url":null,"abstract":"<p><p>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.,<sup>1</sup> Li et al.,<sup>2</sup> and Yuan et al.<sup>3</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103842"},"PeriodicalIF":1.3000,"publicationDate":"2025-05-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"STAR Protocols","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1016/j.xpro.2025.103842","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0
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.
snf1调控的蛋白激酶2s （SnRK2s）受植物激素脱落酸（ABA）和渗透胁迫激活，控制植物生长和胁迫反应；然而，评估SnRK2活性具有挑战性。在这里，我们提出了一种检测植物中SnRK2活性的方案。我们描述了使用抗磷酸化- s175 - snrk2抗体进行免疫印迹、凝胶内激酶测定、带移测定和免疫沉淀激酶测定的步骤。免疫印迹法和凝胶激酶法适用于评估内源性SnRK2活性，而带移法和免疫沉淀激酶法适用于评估转基因系中标记的SnRK2活性。有关本协议使用和执行的完整细节，请参考Li et al.,1 Li et al.，2和Yuan et al.3。
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