PathScan? Multi-Target HCA Stress and Apoptosis Kit

• 产品编号：CST-7103S      品牌：Cell Signaling Technology       原厂货号：7103S
• 产品分类：生化和细胞分析 > 细胞分析试剂盒 > 细胞凋亡试剂盒
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描述：

PathScan® Multi-Target HCA Stress and Apoptosis Kit 中的每个活化状态的抗体都能识别靶蛋白的指定磷酸化形式。Cleaved Caspase-3 (Asp175) and Cleaved PARP (Asp214) antibodies只能识别它们对应的cleaved 蛋白大片段。该单克隆抗体是通过用与人源p53蛋白丝氨酸（15位点）、SAPK/JNK蛋白苏氨酸（183位点）/酪氨酸（185位点）、p38 MAPK蛋白苏氨酸（180位点）/酪氨酸（182位点）、MAPKAPK-2蛋白苏氨酸（334位点）、 c-Jun丝氨酸（73位点）周围残基的合成磷酸化肽段免疫动物后获得的。该多克隆抗体是通过用人源HSP27蛋白丝氨酸（82位点）、caspase-3蛋白天门冬氨酸（175位点）胺端、PARP蛋白天门冬氨酸（214）位点羧端周围残基相一致的合成磷酸化肽段免疫动物后获得的。该多克隆抗体经蛋白A和肽段亲和层析而纯化。CST的 PathScan® Multi-Target HCA Stress and Apoptosis Kit包含八个一抗，该抗体将细胞应激和凋亡信号通路作为目标。该试剂盒用于阐明通过关键通路节点发生的信号转导，使用的方法是自动成像、激光扫描技术或手控免疫荧光显微镜。该试剂盒为研究者提供了一种快速、简单地选择终结点的方法，选择的末端对后续研究十分有用，无论是大量的筛选工程或是单个的小规模实验。免疫荧光实验中抗体的最佳稀释度是10X。因此该抗体能轻易的被稀释到1X工作浓度，并重悬于各种微孔板或载玻片中。每种抗体提供140 μl，足够加入到96孔板(50 μl 1X每孔)中的24孔或两块96孔板中的一行。细胞应激反应和凋亡涉及到信号通路的复杂联络网，当遇到各种潜在破坏因子（包括UV和gamma射线、化学治疗剂、渗透压休克、炎性细胞因子，以及其他环境应激）时，它能保持细胞体内平衡。在研究疾病过程中，由于疾病状态时这些途径出现潜在的解除管制现象，因此细胞对应激反应的应答方式已经成为一个重要的度量标准。比如，癌细胞能影响这些途径，以促进细胞生长和新陈代谢(1)。一些关键的成员涉及属于有丝分裂原活化的蛋白激酶(MAPK)途径中应激活化的信号转导。应急活化的蛋白激酶/Jun-胺端激酶 (SAPK/JNK)就是这些成员中的一个，该激酶更有可能、优先地被各种环境应激活化(2-7)。SAPK/JNK作为一个二聚体被激活时能转位到细胞核中，从而通过它对如c-Jun这样的转录因子的效应而调节转录过程(4,6)。c-Jun蛋白丝氨酸（63、73位点）被SAPK/JNK磷酸化后就活化，活化的c-Jun蛋白影响一系列的生理功能，包括细胞增殖、分化和凋亡(8)。类似于SAPK/JNK途径，p38 MAPK被各种细胞应激反应活化(9-13)。当p38 MAPK蛋白的苏氨酸（180位点）和酪氨酸（182位点）被磷酸化后，它就能激活MAP激酶活化的蛋白激酶2(MAPKAPK-2)，以及转录因子ATF-2, Max, MEF2 (11-16)。 MAPKAPK-2蛋白苏氨酸（222位点）、丝氨酸（272位点）和苏氨酸（334位点）的磷酸化被认为对它的活化十分重要(17)，MAPKAPK-2蛋白能引起热休克蛋白(HSP) 27丝氨酸（15、78、82位点）的磷酸化(9,18)。HSP27是一个小HSP是一个小HSPs，它能以不同水平结构性表达与各种细胞与组织中。应答应激反应后，HSP27的表达水平会提高几倍，使细胞能对抗各种环境改变(18)。SAPK/JNK 、p38 MAPK途径通过p53肿瘤抑制子蛋白的活化，也有助于对细胞周期检验点的控制，它在细胞应答DNA损伤和其他基因组异常时起着主要作用(19)。p53的活化导致细胞周期捕获和DNA修复或凋亡(20)。应激活化的通路也能控制凋亡蛋白和调节子的转录，因此它在凋亡和细胞生存中扮演重要角色。细胞凋亡是调节性细胞自杀机制，特点是细胞核凝聚、细胞萎缩、胞膜空泡化和DNA破碎(21)。 细胞生存需要主动抑制细胞凋亡，这一过程通过抑制前凋亡因子的表达和促进抗凋亡因子的表达来实现。半胱天冬酶，属于半胱氨酸蛋白酶家族，它是凋亡的核心调节子。Caspases启动子(包括caspase-2, -8, -9, -10, -11, -12)紧密连接于前凋亡信号。一旦它被激活，这些Caspases就分裂、活化下游效应子Caspases(包括caspase-3, -6, -7），从而通过裂解细胞蛋白和特异性天冬酰胺残基而导致凋亡的发生(1)。Caspase-3是凋亡的重要执行者，因为它部分或完全水解许多关键蛋白如细胞核酶poly (ADP-ribose) polymerase (PARP)(22)。人们认为PARP涉及应答环境刺激后的DNA修复(23)。PARP也有助于细胞保存它们的生存能力；PARP的分裂有助于细胞分解，同时也是细胞凋亡的标记(24)。

原厂资料：

Specificity / Sensitivity

Each activation state antibody in the PathScan® Multi-Target HCA Stress and Apoptosis Kit recognizes the indicated phosphorylated form of its target. Cleaved Caspase-3 (Asp175) and Cleaved PARP (Asp214) antibodies recognize only the large fragments of their respective cleaved proteins.

Source / Purification

Monoclonal antibody is produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Ser15 of human p53, Thr183/Tyr185 of human SAPK/JNK, Thr180/Tyr182 of human p38 MAPK, Thr334 of human MAPKAPK-2, or Ser73 of human c-Jun. Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser82 of human HSP27, amino-terminal residues adjacent to Asp175 of human caspase-3, or carboxy-terminal residues surrounding Asp214 of human PARP. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

Description

CST’s PathScan® Multi-Target HCA Stress and Apoptosis Kit contains eight primary antibodies that target cellular stress and apoptotic signaling pathways. 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

Cellular stress and apoptosis involve a complex network of signaling pathways that maintain cellular homeostasis when confronted with a variety of potentially damaging effectors, including UV and gamma radiation, chemotherapeutic agents, osmotic shock, inflammatory cytokines, and other environmental stresses. The manner in which cells respond to stress has become an important metric in the study of disease due to the potential deregulation of these pathways in disease states. For example, cancer cells can affect these pathways to promote cell growth and metastasis (1). Some of the key members involved in stress-activated signaling belong to the mitogen-activated protein kinase (MAPK) pathway. The stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK) is one such member that is potently and preferentially activated by a variety of environmental stresses (2-7). SAPK/JNK, when active as a dimer, can translocate to the nucleus where it regulates transcription through its effects on transcription factors such as c-Jun (4,6). Activation of c-Jun by phosphorylation at Ser63 and Ser73 through SAPK/JNK affects a diverse array of biological functions including cell proliferation, differentiation, and apoptosis (8). Similar to the SAPK/JNK pathway, p38 MAPK is activated by a variety of cellular stresses (9-13). When phosphorylated at Thr180 and Tyr182, p38 MAPK has been shown to activate MAP kinase-activated protein kinase 2 (MAPKAPK-2) and the transcription factors ATF-2, Max, and MEF2 (11-16). Phosphorylation at Thr222, Ser272, and Thr334 appears to be essential for the activity of MAPKAPK-2 (17), which can result in the phosphorylation of heat shock protein (HSP) 27 at Ser15, Ser78, and Ser82 (9,18). HSP27 is one of the small HSPs that are constitutively expressed at different levels in various cell types and tissues. In response to stress, the expression level of HSP27 increases several-fold to confer cellular resistance to the adverse environmental change (18). The SAPK/JNK and p38 MAPK pathways also contribute to cell cycle checkpoint control through the activation of the p53 tumor suppressor protein, which plays a major role in cellular response to DNA damage and other genomic aberrations (19). Activation of p53 can lead to either cell cycle arrest and DNA repair or apoptosis (20). Stress-activated pathways also control the transcription of apoptotic proteins and mediators, thereby playing an important role in apoptosis and cell survival. Apoptosis is a regulated cellular suicide mechanism characterized by nuclear condensation, cell shrinkage, membrane blebbing, and DNA fragmentation (21). Cell survival requires the active suppression of apoptosis, which is accomplished by inhibiting the expression of pro-apoptotic factors as well as promoting the expression of anti-apoptotic factors. Caspases, a family of cysteine proteases, are the central regulators of apoptosis. Initiator caspases (including caspase-2, -8, -9, -10, -11, and -12) are closely coupled to pro-apoptotic signals. Once activated, these caspases cleave and activate downstream effector caspases (including caspase-3, -6, and -7), which in turn execute apoptosis by cleaving cellular proteins following specific asparagine residues (1). Caspase-3 is a critical executioner of apoptosis, as it is either partially or totally responsible for the proteolytic cleavage of many key proteins such as the nuclear enzyme poly (ADP-ribose) polymerase (PARP) (22). PARP appears to be involved in DNA repair in response to environmental stress (23). PARP helps cells to maintain their viability; cleavage of PARP facilitates cellular disassembly and serves as a marker of cells undergoing apoptosis (24).

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.