PathScan? Multi-Target HCA DNA Damage Kit

• 产品编号：CST-7101S      品牌：Cell Signaling Technology       原厂货号：7101S
• 产品分类：生化和细胞分析 > 其它细胞分析试剂盒 > 其它细胞分析试剂盒 > 其它
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描述：

PathScan® Multi-Target HCA DNA Damage Kit中的每个激活状态抗体均能识别其特定位点磷酸化的靶蛋白。p21 Waf1/Cip1 antibody能够独立于其磷酸化状态识别p21总蛋白。.单克隆抗体是由合成的人源的针对p53蛋白丝氨酸（15位）的磷酸化肽段、p38 MAPK蛋白的苏氨酸（180位）/酪氨酸（182位）的磷酸化肽段、Chk1蛋白的丝氨酸（345位）磷酸化肽段、SAPK/JNK蛋白的苏氨酸（183位）/酪氨酸（185位）磷酸化肽段、H2A.X蛋白丝氨酸（139位）磷酸化肽段、cdc2蛋白酪氨酸（15位）磷酸化肽段或p21蛋白羧基末端肽段免疫动物生产的。多克隆抗体是由合成的人源的针对Chk2蛋白苏氨酸（68位）的磷酸化肽段免疫动物，采用蛋白A和多肽亲和层析技术纯化生产的。CST’的 PathScan® Multi-Target HCA DNA Damage Kit包含8种检测DNA损伤细胞信号通路的靶蛋白的一抗。该试剂盒为使用自动影像分析、激光扫描平台或人工免疫荧光显微镜分析阐明主要通路节点中信号而设计。该试剂盒提供给研究者一个快速而简捷的方法以选择对后续实验最明显和最有用的终点，包括大型高含量/高通量筛选项目和单个小规模实验。试剂盒中的抗体以其在免疫荧光染色中最佳稀释浓度的10x缓冲液形式提供，从而使得抗体能够易于稀释为其1x工作浓度并分装至多空板或切片。每种抗体提供140 μl(50 μl 1X 每孔)，足够96孔板中24孔或2个96孔板各一行。细胞周期控制涉及一个有序的系列细胞信号事件，旨在保持细胞和生物学途径的完整性和正常的功能。细胞进化出复杂的机制，统称为DNA损伤检验点，调节细胞周期进程来应答基因组损害。由内部或外部因素如紫外线、电离辐射、遗传毒性药物等引起的DNA损伤引发级联事件，阻止细胞周期进程，以允许有时间来修复受损的DNA，如果发生太多的损害则激活细胞死亡途径。监视DNA损伤途径中的信号转导成分是重要的，以帮助理解异常细胞信号与诸如癌症的疾病状态中的未检查的细胞周期控制的关系。DNA损伤检验点可在G1 / S转换期， S期或G2/M期影响细胞周期进程。在G2/M期，cdc2/cyclin B的活性可充当细胞进入有丝分裂的主要调节子(1)。Cdc25C磷酸酶去除抑制cdc2介导的14位苏氨酸和15位酪氨酸磷酸化，允许cdc2/cyclin B最大程度的激活(1,2)。当DNA损伤时，一个信号级联反应被激活，从而抑制Cdc25C激活cdc2/cyclin B的能力。该信号转导通路中的一个邻近事件是Ser139磷酸化的组蛋白H2A.X向亚核灶DNA损伤位点聚集，激活DNA损伤反应核心介质-ATM / ATR激酶。ATM首先被DNA双链断裂激活(4,5)，而ATR被各种DNA损伤和应激激活(6)。ATM / ATR依次启动两个平行的级联反应来灭活cdc2/cyclin B复合体(7)。第一级联迅速抑制通过激活Chk激酶(Chk1对应ATR和Chk2对应ATM)抑制有丝分裂的进程，Chk激酶磷酸化并灭活CDC25，防止cdc2/cyclin B的激活(8-10)。更长期的第二级联参与肿瘤抑制蛋白p53的磷酸化，通过调节p53的下游效应器如肿瘤抑制蛋白p21 Waf1/Cip1 (11)从而引起细胞周期停滞和DNA修复或细胞凋亡。DNA损伤时，p53的一系列位点发生磷酸化并通过p53基因反应元件上调p21转录。 p21通过抑制细胞周期依赖性蛋白激酶包括cdc2一个子集可以阻止细胞周期进程(12,13)。除了典型的ATM/ATR检验点信号，SAPK/JNK和p38 MAP激酶途径也被多种细胞应激激活，包括炎症性细胞因子、紫外线和生长因子(14,15)。这些应激激活途径有助于G2/M期经由激活p53和其它MAPK底物的检验点控制，如MAPKAPK-2，它可以直接影响检验点的级联反应组件如cdc25(8)。

原厂资料：

Specificity / Sensitivity

Each activation state antibody in the PathScan® Multi-Target HCA DNA Damage Kit recognizes the indicated phosphorylated form of its target. p21 Waf1/Cip1 antibody recognizes total p21 protein, independent of its phosphorylation state.

Source / Purification

Monoclonal antibody is produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Ser15 of human p53, Thr180/Tyr182 of human p38 MAPK, Ser345 of human Chk1, Thr183/Tyr185 of human SAPK/JNK, Ser139 of human histone H2A.X, Tyr15 of human cdc2, or the carboxy terminus of human p21. Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr68 of human Chk2. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

Description

CST’s PathScan® Multi-Target HCA DNA Damage Kit contains eight primary antibodies that target the DNA damage cellular signaling pathway. This kit is designed to elucidate the signaling occurring through key pathway nodes using automated imaging or laser scanning platforms or manual immunofluorescent microscopy. The kit provides the investigator with a quick and easy means to choose the endpoints that will be the most robust and useful for subsequent studies, whether large high content/high throughput screening projects or single small-scale experiments. The antibodies are supplied at 10X of their optimal dilution for immunofluorescent applications. This allows the antibodies to be easily diluted to their 1X working concentrations and dispensed into multi-well plates or slides. 140 μl of each antibody is supplied, which is sufficient for 24 wells on 96-well plates (50 μl 1X per well) or one row on two 96-well plates.

Background

Cell cycle control involves an ordered series of cellular signaling events designed to maintain the integrity and proper function of cells and biological pathways. Cells have evolved complex mechanisms, collectively termed the DNA damage checkpoint, to modulate cell cycle progression in response to genomic insult. Damage to DNA caused by either internal or external sources such as UV light, ionizing radiation, genotoxic agents, etc. initiates a cascade of events that blocks cell cycle progression to either allow time to repair damaged DNA, or activate cell death pathways if too much damage has been incurred. Monitoring the signaling components of the DNA damage pathway is important to aid in understanding the aberrant cellular signaling associated with unchecked cell cycle control in disease states such as cancer. Cell cycle progression can be affected by the DNA damage checkpoint at the G1/S transition, during S phase, or at the G2/M phase transition. At G2/M, cdc2/cyclin B activity acts as a master regulator of entry into mitosis (1). The cdc25C phosphatase removes inhibitory phosphorylation on Thr14 and Tyr15 of cdc2, allowing for maximal activation of cdc2/cyclin B (1,2). When DNA damage occurs, a signaling cascade is activated that inhibits the ability of cdc25 to activate cdc2/cyclin B. A proximal event in the signaling pathway is localization of Ser139-phosphorylated histone H2A.X to sites of DNA damage at subnuclear foci (3). Histone H2A.X and other mediators recruit additional signaling molecules to the site of the damage, activating the ATM/ATR kinases, the central mediators of the DNA damage response. ATM is primarily activated by double strand breaks of DNA (4,5), while ATR is activated by a variety of DNA lesions and replication stresses (6). ATM/ATR in turn initiate two parallel cascades that inactivate the cdc2/cyclin B complex (7). The first cascade rapidly inhibits progression into mitosis through the activation of the Chk kinases (Chk1 for ATR and Chk2 for ATM), which phosphorylate and inactivate cdc25, preventing activation of cdc2/cyclin B (8-10). The more long-term second cascade involves phosphorylation of the tumor suppressor protein p53, leading to either cell cycle arrest and DNA repair or apoptosis through regulation of p53 downstream effectors, including the tumor suppressor protein p21 Waf1/Cip1 (11). Upon DNA damage, p53 is phosphorylated at a number of sites and up-regulates p21 transcription via a p53 responsive element. p21 expression can block cell cycle progression by inhibiting a subset of the cyclin-dependent kinases including cdc2 (12,13). In addition to canonical ATM/ATR checkpoint signaling, the SAPK/JNK and p38 MAP kinase pathways are activated by a variety of cellular stresses including inflammatory cytokines, UV light, and growth factors (14,15). These stress-activated pathways contribute to G2/M checkpoint control through activation of p53 and other MAPK substrates, such as MAPKAPK-2, which can directly affect components of the checkpoint cascade, such as cdc25 (8). 

注意事项：

Storage: Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM
NaCl, 100 μg/ml BSA and 50% glycerol. Store at –20°C.
Do not aliquot the antibodies.