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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