| Genome-Wide siRNA Screen for Anti-Cancer Drug Resistance in Adherent Cell Lines
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Author:
Date:
2015-05-20
[Abstract] The expression of genes is frequently manipulated in cell lines to study their cellular functions. The use of exogenous small Interfering RNAs (siRNAs) is a very efficient technique to temporarily downregulate the expression of genes of interest [reviewed by Hannon and Rossi (2004)]. A genome-wide siRNA library allows the user to study both the effect of each individual gene on a particular cell phenotype in a high throughput manner and also assess its phenotypic effect relative to all other genes targeted. Several factors that potentially influence the outcome of a screen need to be considered when performing a large siRNA screen (Jiang et al., 2011). Here we present a detailed protocol for a genome-wide screen to identify genes involved in anti-cancer drug resistance using the ...
[摘要] 基因的表达经常在细胞系中操作以研究它们的细胞功能。外源小干扰RNA（siRNA）的使用是临时下调感兴趣的基因的表达的非常有效的技术[由Hannon和Rossi（2004）综述]。全基因组siRNA文库允许用户以高通量方式研究每个个体基因对特定细胞表型的作用，并且还评估其相对于所有其他靶向基因的表型效应。当进行大siRNA筛选时，需要考虑潜在地影响筛选结果的几个因素（Jiang等人，2011）。在这里，我们提出了一个详细的协议，使用人类siGENOME库从Dharmacon的基因组范围屏幕识别参与抗癌耐药性的基因。在该方案中，我们关注对表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKI）厄洛替尼在对EGFR-TKIs敏感的肺癌细胞系PC9中的治疗的抗性（de Bruin等。，2014）。该方案可用于其他细胞系和其他药物治疗，我们在下面的注释中扩展。
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| Ki67 Immunofluorescence on Bovine Cell Lines
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Author:
Date:
2013-11-05
[Abstract] This is a rapid protocol to test the effects of drugs treatment on bovine cell replication using Ki67 staining. Ki67 is associated with cell proliferation and is present during all active phases of the cell cycle (G1, S, G2, and mitosis), but is absent from resting cells (G0).
[摘要] 这是一个快速协议，以测试药物治疗对牛细胞复制使用Ki67染色的影响。 Ki67与细胞增殖相关，并且在细胞周期（G1，S，G2和有丝分裂）的所有活性期期间存在，但在静息细胞（G0）中不存在。
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| β1 Integrin Cell-surface Immunoprecipitation (Selective Immunoprecipitation)
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Author:
Date:
2013-11-05
[Abstract] Immunoprecipitation (IP) is a widely used method to isolate a specific protein from a mixed protein sample using an antibody that exclusively binds to that particular protein. This technique allows studying protein-protein and protein-nucleic acid interactions or to identify post-translational protein modifications. Many proteins, in particular cell surface receptors, localize to different compartments within cells where they elicit distinct functions by interacting with specific proteins. Integrins represent a major family of cell surface receptors consisting of non-covalently associated α and β subunits that mediate the interaction of cells with their environment. However, integrins do not only localize to the cell surface but are also present in other compartments including the ...
[摘要] 免疫沉淀（IP）是一种广泛使用的方法，使用仅与该特定蛋白结合的抗体从混合蛋白样品中分离特定蛋白。这种技术允许研究蛋白质 - 蛋白质和蛋白质 - 核酸相互作用或识别翻译后蛋白质修饰。许多蛋白质，特别是细胞表面受体，定位于细胞内的不同区室，其中它们通过与特定蛋白质相互作用引起不同的功能。整联蛋白代表由非共价缔合的α和β亚基组成的细胞表面受体的主要家族，其介导细胞与其环境的相互作用。然而，整联蛋白不仅定位于细胞表面，而且存在于其它区室中，包括内质网和内体，其中它们与不同组的相互作用配偶体接合或显示不同的翻译后修饰。来自细胞裂解物的β1整联蛋白的标准免疫沉淀，没有预先分级分离来自所有区室的β1整联蛋白。相比之下，细胞表面β1整合素的选择性免疫沉淀允许富集β1整联蛋白在细胞表面上的库，从而使来自其他亚细胞区室的β1整联蛋白的污染最小化。为了实现这一点，活细胞与β1整合素特异性抗体在冰上孵育以在细胞裂解和沉淀之前标记细胞表面β1整联蛋白。
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