Protein immunolocalization in maize tissues.
Protein immunolocalization in maize tissues.
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DOI：
10.1007/978-1-62703-221-6_14
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发表时间：
2013-01-01
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影响因子：
--
通讯作者：
Varotto, Serena
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文献类型：
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作者：
Forestan, Cristian;Carraro, Nicola;Varotto, Serena
在转录水平和蛋白质水平上分析基因表达在植物发育生物学中具有重要意义。蛋白质可以通过使用特异性抗体的免疫定位或产生携带与荧光蛋白融合的特异性蛋白质的报告细胞系来以亚细胞分辨率定位。免疫定位特别适合于确认转基因报告细胞系的表达模式。它还代表了一种有效的替代方案，特别是对于转化耗时且仍然经常不成功的植物如玉米，绕过了融合蛋白的一些副作用。事实上，特异性抗体的免疫定位和共聚焦显微镜下观察玉米组织的可用性是一个强大的工具，研究蛋白质靶向不同cellularcompartment.In以下章节中，我们描述了完整的程序定位蛋白质在不同的玉米组织在细胞和亚细胞水平，使用多克隆或单克隆抗体。根据组织和所需的用途：落射荧光或共聚焦显微镜观察，可以将组织包埋在不同的基质中，例如paraplast、PEG 400和琼脂糖。一个附加的协议，用于分析的核分布的修饰组蛋白在压扁玉米根尖。
The analysis of gene expression at transcript and at protein level is of outstanding importance in plant developmental biology. Proteins can be localized with subcellular resolution by immunolocalization using specific antibodies or generating reporter lines carrying the specific protein fused to a fluorescent protein. Immunolocalization is particularly suitable to confirm the expression pattern of transgenic reporter lines. It also represents a valid alternative, especially for plants such as maize, for which transformation is time consuming and still often unsuccessful, by-passing also some side-effects of fusion proteins. Indeed, the availability of specific antibodies for immunolocalizations and observations of maize tissues under a confocal microscope is a powerful tool for studying protein targeting to different cellular compartments.In the following chapter we describe the complete procedure for the localization of proteins in different maize tissues both at cellular and sub-cellular level, using polyclonal or monoclonal antibodies. Tissues can be embedded in different substrates, such as paraplast, PEG400 and agarose, depending on the tissue and the desired use: epifluorescence or confocal microscope observations. An additional protocol for the analysis of the nuclear distribution of modified histones in squashed maize root apexes is also presented.