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IGEPAL® CA-630

IGEPAL CA-630

Company: Sigma-Aldrich
Catalog#: I3021
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Active Cdk5 Immunoprecipitation and Kinase Assay
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 preserve both p35- and p39-Cdk5 complexes to assess total Cdk5 activity. Cells are lysed and protein concentration is determined in the post-nuclear supernatant. Cdk5 is immunoprecipitated from equal ... [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; ...

Flow Cytometric Analysis of HIV-1 Transcriptional Activity in Response to shRNA Knockdown in A2 and A72 J-Lat Cell Lines
Author:
Date:
2017-06-05
[Abstract]  The main obstacle to eradicating HIV-1 from patients is post-integration latency (Finzi et al., 1999). Antiretroviral treatments target only actively replicating virus, while latent infections that have low or no transcriptional activity remain untreated (Sedaghat et al., 2007). To eliminate viral reservoirs, one strategy focuses on reversing HIV-1 latency via ‘shock and kill’ (Deeks, 2012). The basis of this strategy is to overcome the molecular mechanisms of HIV-1 latency by therapeutically inducing viral gene and protein expression under antiretroviral therapy and to cause selective cell death via the lytic properties of the virus, or the immune system now recognizing the infected cells. Recently, a number of studies have described the therapeutic potential of ... [摘要]  消除HIV-1患者的主要障碍是后整合延迟(Finzi等人,1999)。抗逆转录病毒治疗仅针对主动复制病毒,而具有低转录活性或无转录活性的潜伏感染仍未得到治疗(Sedaghat等人,2007)。为了消除病毒性水库,一项战略重点是通过“休克和杀死”来逆转HIV-1潜伏期(Deeks,2012)。该策略的基础是通过在抗逆转录病毒治疗下通过治疗性诱导病毒基因和蛋白质表达来克服HIV-1潜伏期的分子机制,并通过病毒的溶解性质或现在识别感染细胞的免疫系统引起选择性细胞死亡。最近,许多研究已经描述了药物抑制人类溴结构域蛋白质的溴结构域和末端(BET)家族的成员的治疗潜力(Filippakopoulos等人,2010; Dawson等人& / em>,2011; Delmore等人,2011),其包括BRD2,BRB3,BRD4和BRDT。小分子BET抑制剂,例如JQ1(Filippakopoulos et al。,2010; Delmore等人,2011),I-BET(Nicodeme等人< / ...

Polysome Analysis
Author:
Date:
2017-03-20
[Abstract]  Polysome analysis is a method to separate mRNAs from a cell into actively translating and non-translating fractions depending on their association with polysomes. By this protocol, cell lysates are fractionated by sucrose density gradient ultracentrifugation. Free mRNA fraction and various ribosomal fractions, such as 40S, 60S, monosomes and polysomes are collected by fractionation. Association of particular mRNAs with these fractions is detected by reverse transcription – PCR to investigate the translational state of the mRNA. [摘要]  多聚赖氨酸分析是一种将mRNA从细胞分离为主动翻译和非翻译部分的方法,这取决于它们与多核糖体的关联。通过该方案,通过蔗糖密度梯度超速离心分离细胞裂解物。通过分级收集游离mRNA级分和各种核糖体级分,例如40S,60S,单体和多核糖体。通过逆转录PCR检测特异性mRNA与这些级分的关联,以研究mRNA的翻译状态。

背景 细胞mRNA在任何时间点分布到主动翻译和非翻译池中,并可响应于各种刺激而在这些池之间动态地重新分布。主动翻译的mRNA具有较高数量的与它们相关的核糖体,与mRNA相关的核糖体数量是mRNA的翻译状态的量度。因此,从细胞中分离核糖体时,会在多核糖体组分中发现主要转录的mRNA,而在游离mRNA部分或与40S核糖体亚基相关的非翻译/不良翻译mRNAs。因此,多聚赖氨酸分析是根据其与多核糖体的关联将mRNA从细胞分离为主动翻译和非翻译部分的方法(Ray et al。,2009; Poria等人, >。,2016)。可以通过RT-PCR检测单个mRNA与翻译/非翻译级分的关联,而通过RNA测序或微阵列分析可以鉴定mRNA的整个翻译或非翻译池。

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