O'Hagan Robert, Topalidou Irini
Biology Department, Montclair State University, Montclair, USA.
Department of Biochemistry, University of Washington, Seattle, Washington, USA.
Bio Protoc. 2021 Oct 5;11(19):e4172. doi: 10.21769/BioProtoc.4172.
Although the advent of genetically-encoded fluorescent markers, such as the green fluorescent protein (GFP; Chalfie , 1994 ), has enabled convenient visualization of gene expression , this method is generally not effective for detecting post-translational modifications because they are not translated from DNA sequences. Genetically-encoded, fluorescently-tagged transgene products can also be misleading for observing expression patterns because transgenes may lack endogenous regulatory DNA elements needed for precise regulation of expression that could result in over or under expression. Fluorescently-tagged proteins created by CRISPR genome editing are less prone to defective expression patterns because the loci retain endogenous DNA elements that regulate their transcription (Nance and Frøkjær-Jensen, 2019). However, even CRISPR alleles encoding heritable fluorescently-tagged protein markers can result in defects in function or localization of the gene product if the fluorescent tag obstructs or otherwise interferes with important protein interaction domains or affects the protein structure. Indirect immunofluorescence is a method for detecting endogenous gene expression or post-translational modifications without the need for transgenesis or genome editing. Here, we present a reliable protocol in which nematodes are fixed, preserved, and permeabilized for staining with a primary antibody to bind proteins or post-translational modifications, which are then labeled with a secondary antibody conjugated to a fluorescent dye. Use of this method may be limited by the availability of (or ability to generate) a primary antibody that binds the epitope of interest in fixed animals. Thousands of animals are simultaneously subjected to a series of chemical treatments and washes in a single centrifuge tube, allowing large numbers of identically-treated stained animals to be examined. We have successfully used this protocol (O' Hagan , 2011 and 2017; Power , 2020 ) to preserve and detect post-translational modifications of tubulin in ciliated sensory neurons and to detect non-modified endogenous protein (Topalidou and Chalfie, 2011).
尽管诸如绿色荧光蛋白（GFP；Chalfie，1994）等基因编码荧光标记的出现，使得基因表达的可视化变得便捷，但这种方法通常对检测翻译后修饰无效，因为它们并非从DNA序列翻译而来。基因编码的、荧光标记的转基因产物在观察表达模式时也可能产生误导，因为转基因可能缺乏精确调控表达所需的内源调控DNA元件，这可能导致表达过度或不足。由CRISPR基因组编辑产生的荧光标记蛋白不太容易出现有缺陷的表达模式，因为这些位点保留了调控其转录的内源DNA元件（Nance和Frøkjær-Jensen，2019）。然而，如果荧光标签阻碍或以其他方式干扰重要的蛋白质相互作用结构域或影响蛋白质结构，即使是编码可遗传荧光标记蛋白标记的CRISPR等位基因也可能导致基因产物的功能或定位缺陷。间接免疫荧光是一种无需转基因或基因组编辑即可检测内源基因表达或翻译后修饰的方法。在此，我们提出一种可靠的方案，其中线虫被固定、保存并通透处理，以便用一抗染色以结合蛋白质或翻译后修饰，然后用与荧光染料偶联的二抗进行标记。这种方法的使用可能受到能够结合固定动物中感兴趣表位的一抗的可用性（或产生能力）的限制。数千只动物在单个离心管中同时接受一系列化学处理和洗涤，从而可以检查大量经过相同处理的染色动物。我们已成功使用该方案（O'Hagan，2011年和2017年；Power，2020年）来保存和检测纤毛感觉神经元中微管蛋白的翻译后修饰，并检测未修饰的内源蛋白（Topalidou和Chalfie，2011年）。
