Kaufmann Christine, Sauter Margret
Plant Developmental Biology and Plant Physiology, University of Kiel, Am Botanischen Garten 5, 24118, Kiel, Germany.
Methods Mol Biol. 2017;1621:151-159. doi: 10.1007/978-1-4939-7063-6_15.
Plant receptor-like kinases (RLKs) are regulated by posttranscriptional modification and by interaction with regulatory proteins. A common modification of RLKs is (auto)phosphorylation, and a common regulatory protein is the calcium sensor calmodulin (CaM). We have developed protocols to detect the interaction of an RLK with CaM. The interaction with CaM was shown by bimolecular fluorescence complementation (BiFC) (see Chapter 14) and pull-down assay (this chapter). Both methods offer unique advantages. BiFC is useful in showing interaction of soluble as well as of membrane-bound proteins in planta. Pull-down assays are restricted to soluble proteins and provide in vitro data. The pull-down assay provides the advantage that proteins can be modified prior to binding and that experimental conditions such as the concentration of Ca or other divalent cations can be controlled. This chapter provides a pull-down protocol to study RLK-CaM interaction with optional steps to investigate the impact of RLK phosphorylation or of Ca.
植物类受体激酶（RLKs）受转录后修饰以及与调节蛋白相互作用的调控。RLKs常见的修饰是（自）磷酸化，常见的调节蛋白是钙传感器钙调蛋白（CaM）。我们已经开发出检测RLK与CaM相互作用的方法。通过双分子荧光互补（BiFC）（见第14章）和下拉试验（本章）证明了与CaM的相互作用。这两种方法都有独特的优势。BiFC有助于显示植物中可溶性蛋白以及膜结合蛋白的相互作用。下拉试验仅限于可溶性蛋白，并提供体外数据。下拉试验的优点是蛋白质在结合前可以进行修饰，并且可以控制诸如钙或其他二价阳离子浓度等实验条件。本章提供了一个下拉试验方案，用于研究RLK-CaM相互作用，并带有研究RLK磷酸化或钙影响的可选步骤。
