{{'Search' | translate}}
 

100-mm tissue culture Petri dish

Falcon 100mm TC处理的细胞培养皿

Company: Corning
Catalog#: 353003
Bio-protocol()
Company-protocol()
Other protocol()

Notch Ligand Binding Assay Using Flow Cytometry
Author:
Date:
2017-12-05
[Abstract]  Notch signaling is an evolutionarily conserved signaling pathway that plays an indispensable role during development, and in the maintenance of homeostatic processes, in a wide variety of tissues (Kopan, 2012; Hori et al., 2013). The multifaceted roles of Notch signaling are stringently regulated at different levels. One of the most important aspects of regulation is the binding of different Notch ligands to each Notch receptor (NOTCH1-NOTCH4). Canonical ligands Delta or Serrate (in Drosophila), and Delta-like (DLL1 and DLL4) or Jagged (JAG1 and JAG2) (in mammals), are transmembrane glycoproteins. Ligands expressed on one cell bind to Notch receptors on an adjacent cell to induce Notch signaling. Glycosylation of Notch receptor extracellular domain by O-fucose and ... [摘要]  Notch信号传导是进化上保守的信号传导途径,在发育过程中以及在各种组织中维持体内平衡过程中起着不可或缺的作用(Kopan,2012; Hori等人,2013)。 Notch信号的多方面作用在不同的层面上受到严格的调控。监管的最重要的方面之一是不同Notch配体与各Notch受体(NOTCH1-NOTCH4)的结合。典型的配体Delta或Serrate(在果蝇中)和Delta样(DLL1和DLL4)或Jagged(JAG1和JAG2)(在哺乳动物中)是跨膜糖蛋白。在一个细胞上表达的配体结合相邻细胞上的Notch受体以诱导Notch信号传导。通过O-岩藻糖和O-GlcNAc聚糖对Notch受体细胞外结构域的糖基化作为Notch信号强度的关键调节剂已经得到很好的确立(Stanley and Okajima,2010; Haltom and Jafar-Nejad,2015; Sawaguchi et al。 >,2017)。为了表征Notch配体与分离细胞中的Notch受体的结合,我们利用人Fc区在C端标记的Notch配体胞外结构域,并通过流式细胞术确定荧光抗Fc抗体的结合。
【背景】众所周知,细胞增殖,分化和凋亡受Notch信号调节。 ...

Knock-in Blunt Ligation Utilizing CRISPR/Cas9
Author:
Date:
2017-03-05
[Abstract]  The incorporation of the CRISPR/Cas9 bacterial immune system into the genetic engineering toolbox has led to the development of several new methods for genome manipulation (Auer et al., 2014; Byrne et al., 2015). We took advantage of the ability of Cas9 to generate blunt-ended double-strand breaks (Jinek et al., 2012) to introduce exogenous DNA in a highly precise manner through the exploitation of non-homologous end-joining DNA repair machinery (Geisinger et al., 2016). This protocol has been successfully applied to traditional immortalized cell lines and human induced pluripotent stem cells. Here we present a generalized protocol for knock-in blunt ligation, using HEK293 cells as an example. [摘要]  将CRISPR / Cas9细菌免疫系统并入基因工程工具箱已经导致了几种用于基因组操作的新方法的开发(Auer等人,2014; Byrne等人,2015)。我们利用Cas9产生平端双链断裂的能力(Jinek等人,2012),以高度精确的方式通过开发非同源末端引物来引入外源DNA,加入DNA修复机械(Geisinger等人,2016)。该方案已成功应用于传统的永生化细胞系和人诱导多能干细胞。在这里,我们提出了使用HEK293细胞作为例子的敲入钝性连接的一般化方案。

背景 当我们概念化敲门钝性结扎(Geisinger等人,2016)时,开发用于CRISPR / Cas9的绝大多数方法都集中在提高同源重组的效率。然而,有一个例外:在斑马鱼中开发的同源性独立的基于质粒的敲入方法(Auer等人,2014)。这种方法,如敲入钝性连接,依赖于典型的非同源末端连接的机制,以线性化的,平端的双链DNA片段以高精度插入到基因组双链断裂中,核苷酸损失最小。这两种方法类似于为锌指核酸酶和被称为专性连接门控重组的TALEN开发的方法(ObLiGaRe; Maresca等人,2013),其依赖于产生相容的突出端以促进将靶DNA插入基因组。 ...

MPM-2 Mediated Immunoprecipitation of Proteins Undergoing Proline-directed Phosphorylation
Author:
Date:
2016-12-05
[Abstract]  Immunoprecipitation (IP) represents a widely utilized biochemical method to isolate a specific protein from a complex mixture taking advantage of an antibody that specifically recognizes that particular target molecule. This procedure is extremely versatile and can be applied to concentrate a specific protein, to identify interacting partners in complex with it or to detect post-translational modifications. The mitotic protein monoclonal 2 (MPM-2) is an antibody originally raised against extracts of synchronized mitotic HeLa cells to identify proteins selectively present in mitotic, and not in interphase-cells (Davis et al., 1983). MPM-2 recognizes phosphorylated serine or threonine residues followed by proline (pS/T-P), consensus epitopes generated by the concerted action of ... [摘要]  免疫沉淀(IP)代表广泛使用的生物化学方法,以利用特异性识别特定靶分子的抗体从复杂混合物中分离特异性蛋白质。该程序是非常通用的,可以应用于集中特定蛋白质,识别与其复合的相互作用的配偶体或检测翻译后修饰。有丝分裂蛋白单克隆2(MPM-2)是最初针对同步有丝分裂HeLa细胞的提取物产生的抗体,以鉴定选择性存在于有丝分裂中的蛋白质,而不是在间期细胞中(Davis等,1983)。 MPM-2识别磷酸化的丝氨酸或苏氨酸残基,随后是脯氨酸(pS / T-P),由脯氨酸指导的蛋白激酶和磷酸酶的共同作用产生的共有表位(Lu等,2002)。这些可逆磷酸化事件已经出现以通过促进目标上的构象变化来控制各种细胞过程,这不仅仅是由于磷酸化事件本身。这些基序一旦被磷酸化,就能够招募Pin1(肽基 - 脯氨酰异构酶NIMA相互作用蛋白1)(Lu等人,1996; Lu和Zhou,2007),这是一个促进肽键上的顺式/反式异构化反应的伴侣,在基础功能不同的构象之间切换底层(Lu,2004; Wulf等人,2005)。该方案描述了使用支架分子突触后密度蛋白-95(PSD-95)(Chen等人,2005),神经元Pin1靶(Antonelli等,2016)作为示例的基于MPM-2的免疫沉淀策略以说明详细的程序。
【背景】由MPM-2抗体识别的抗原的鉴定代表了发现经过磷酸化脯氨酰异构化调控机制的靶分子的有用起点。 ...

Comments