Active Cdk5 Immunoprecipitation and Kinase Assay
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Author:
Date:
2017-07-05
[Abstract] Cdk5 activity is regulated by the amounts of two activator proteins, p35 and p39 (Tsai et al., 1994; Zheng et al., 1998; Humbert et al., 2000). The p35-Cdk5 and p39-Cdk5 complexes have differing sensitivity to salt and detergent concentrations (Hisanaga and Saito, 2003; Sato et al., 2007; Yamada et al., 2007; Asada et al., 2008). Cdk5 activation can be directly measured by immunoprecipitation of Cdk5 with its bound activator, followed by a Cdk5 kinase assay. In this protocol, buffers for cell lysis and immunoprecipitation are intended to ...
[摘要] Cdk5活性受两种激活蛋白p35和p39(Tsai et al。,1994; Zheng et al。,1998; Humbert等人)的量的调节,2000)。 p35-Cdk5和p39-Cdk5复合物对盐和洗涤剂浓度的敏感性不同(Hisanaga和Saito,2003; Sato et al。,2007; Yamada等人, 2007; Asada 等人,2008)。 Cdk5激活可以通过Cdk5与其结合的激活剂的免疫沉淀直接测量,随后进行Cdk5激酶测定。在该方案中,用于细胞裂解和免疫沉淀的缓冲液旨在保持p35-和p39-Cdk5复合物以评估总Cdk5活性。裂解细胞,并在核后上清液中测定蛋白浓度。 Cdk5在实验组之间从等量的总蛋白免疫沉淀。然后进行洗涤以除去外来蛋白质并平衡激酶缓冲液中的Cdk5-活化剂复合物。然后将Cdk5与组蛋白H1孵育,组蛋白H1是Cdk5和[γ- 32 P] ATP在体外成功建立的靶标。反应通过SDS-PAGE解析并转移到膜上,用于可视化H1磷酸化和免疫沉淀的Cdk5水平的免疫印迹。我们已经使用该测定来建立p39作为少突神经胶质谱系中Cdk5的主要活化剂。然而,该测定法适用于对裂解条件进行适当调整的其它细胞谱系或组织。 【背景】虽然Cdk5通常与神经元功能相关,但最近的工作已经证明Cdk5也可以调节少突胶质细胞祖细胞(OPC)的发育(Tang等人,1998; ...
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Detection of Mitogen-activated Protein Kinase (MAPK) Activation upon Exogenous Chemical Application in Arabidopsis Protoplasts
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Author:
Date:
2015-09-20
[Abstract] The mitogen activated protein kinase cascade is a highly conserved signal pathway in plants. The exogenous chemicals, like hormones, can trigger a series of signalling cascades, including MAPK pathway, to modulate the plant physiology. Upon activation, some MAPKs are phosphorylated. It is important to develop methods that can detect changes in the phosphorylation status of MAPKs in plants when they come in contact with external chemicals. This method describes the exogenous treatment of Arabidopsis protoplasts with Kinetin and subsequent detection of the activated MAPKs. This method ...
[摘要] 丝裂原活化蛋白激酶级联是植物中高度保守的信号通路。 外源化学物质,如激素,可以触发一系列信号级联,包括MAPK途径,以调节植物生理。 在激活时,一些MAPK被磷酸化。 开发能够检测植物中MAPK在与外部化学品接触时磷酸化状态的变化的方法是重要的。 该方法描述了用激动素对随后检测激活的MAPK的拟南芥原生质体的外源处理。 该方法可用于研究外源施加的化合物对拟南芥中MAPK信号级联的影响。
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Protein Translation Study – Label Protein with S35 Methionine in Cells
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Author:
Date:
2012-11-05
[Abstract] To follow protein synthesis, cells should be incubated with radioactive amino acid such as [35S] methionine during mRNA translation. Then, the neosynthetized protein will be identified by an autoradiography after immunoprecipitation with a specific antibody and separation on a polyacrylamide denaturing gel.
[摘要] 该实验方案的中文版正在准备中...
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