Ovečka Miroslav, Samajová Olga, Baluška František, Samaj Jozef
Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University Olomouc, Šlechtitelů 11, 783 71, Olomouc, Czech Republic,
Methods Mol Biol. 2014;1171:117-30. doi: 10.1007/978-1-4939-0922-3_10.
Signals of different nature are transduced in cells through signal transduction pathways, where mitogen-activated protein kinases (MAPKs) play an important role as signaling molecules. Views into intracellular localization of MAPKs are critical for the understanding of their spatial and temporal functions, like activation-based relocation, compartmentation, or interactions with local substrates. Localization of MAPKs in cells is thus very useful cell biological approach, extending complex mode of cell signaling characterization in plants. Here, we present a method for subcellular immunofluorescence localization of MAPKs using protein- or phospho-specific antibodies, performed on sectioned fixed plant samples. It is based on embedding of samples in the Steedman's wax, a low-melting point polyester wax embedding medium, which maintains high antigenicity of studied proteins. In addition, exposure of dewaxed sections to antibodies allows for their efficient penetration. Altogether, it makes this simple method a good tool in the efficient subcellular localization of diverse proteins, including plant MAPKs.
不同性质的信号通过信号转导途径在细胞中进行转导，其中丝裂原活化蛋白激酶（MAPKs）作为信号分子发挥着重要作用。了解MAPKs在细胞内的定位对于理解其时空功能至关重要，比如基于激活的重新定位、区室化或与局部底物的相互作用。因此，MAPKs在细胞中的定位是一种非常有用的细胞生物学方法，扩展了植物细胞信号传导特征的复杂模式。在这里，我们介绍一种使用蛋白质特异性或磷酸化特异性抗体对固定的植物切片样本进行MAPKs亚细胞免疫荧光定位的方法。该方法基于将样本嵌入Steedman蜡中，Steedman蜡是一种低熔点聚酯蜡包埋介质，它能保持所研究蛋白质的高抗原性。此外，脱蜡切片与抗体接触可使其有效渗透。总之，这使得这种简单方法成为有效进行包括植物MAPKs在内的多种蛋白质亚细胞定位的良好工具。
