# Protocol for detecting SnRK2 kinase activity in plants by immunoblotting, in-gel assay, band shift, and immunoprecipitation

## TL;DR

This paper provides a detailed protocol for detecting SnRK2 kinase activity in plants using multiple experimental techniques.

## Contribution

A new protocol is introduced for evaluating SnRK2 activity using immunoblotting, in-gel assays, band shift, and immunoprecipitation.

## Key findings

- Immunoblotting and in-gel kinase assays are effective for endogenous SnRK2 activity evaluation.
- Band-shift and immunoprecipitated kinase assays are suitable for tagged SnRK2 variants in transgenic lines.

## 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

•Instructions for evaluating plant SnRK2 kinase activity in vivo•Steps for monitoring SnRK2 activation through immunoblotting and in-gel kinase assays•Applications for both endogenous and tagged SnRK2 variants

Instructions for evaluating plant SnRK2 kinase activity in vivo

Steps for monitoring SnRK2 activation through immunoblotting and in-gel kinase assays

Applications for both endogenous and tagged SnRK2 variants

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

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.