PathScan? Intracellular Signaling Array Kit (Fluorescent Readout)

• 产品编号：CST-7744S      品牌：CST       原厂货号：7744S
• 产品分类：ELISA及其它免疫分析 > ELISpot及FluoroSpot > FluoroSpot试剂盒
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只有当特定残基磷酸化或裂解时，PathScan® Intracellular Signaling Array Kit (Fluorescent Readout)能够检测指示的细胞蛋白和信号节点。（请参见靶点阵列图）。已发现靶点之间没有显著的交叉反应。本试剂盒优化的细胞裂解液，可将总蛋白浓度稀释到0.2至1 mg/ml之间（见试剂盒指南）。所有的捕获抗体已被验证为人和小鼠来源的样品。PathScan® Intracellular Signaling Array Kit (Fluorescent Readout)是一个基于盖玻片的建立在夹心免疫原理之上的抗体阵列。当磷酸化或裂解时，阵列试剂盒可同时检测18个重要特征信号分子。可发现，捕获的靶特异性抗体被复制在硝酸纤维素膜包被的玻璃载玻片上。每个试剂盒包含2张16-pad 盖玻片，从而使用户能够在一次实验中测试32个样品，产生576个数据点。细胞裂解物在载玻片上孵育，其次是用生物素化检测抗体混合物。然后用链霉亲和素耦联的DyLight® 680可视化检测结合的抗体。载玻片上的荧光图像可以通过数字成像系统获取，并用阵列分析软件准确量化。磷酸化和蛋白水解是两个普遍的共价翻译后修饰，在生物学中代表重要的调控机制。检测一组细胞蛋白的这些修饰，在细胞生物学中发挥了很好的理解作用，提供了广阔的细胞内信号的简单印象。MAPK / ERK级联是最好的特征性和广泛研究的信号模块之一。它参与广泛的细胞过程，如增殖、分化和运动。MAPK/ Erk由范围广泛的细胞外信号激活，包括生长因子、细胞因子、激素、神经递质。它被双特异性激酶MEK1和MEK2双磷酸化苏氨酸（202位）和酪氨酸（204位）激活。p38和JNK MAPKs是信号传导部分两个额外结构相关的核心成分。p38和JNK由多种MAPK激酶通过类似的双磷酸化机制激活，与促炎细胞因子、压力条件或遗传毒性相响应。
 Stat1 和Stat3都是参与免疫和炎症的重要信号分子，可以被各种细胞因子或生长因子激活。Stat1 和Stat3 由Jak家族细胞因子受体栓酪氨酸激酶，或者某些情况下的其他酪氨酸激酶，如Src，分别在酪氨酸（701位）和酪氨酸（705位）磷酸化。 Akt是一种蛋白激酶，通常响应发送生长和存活信号的生长因子的刺激。由TORC2复合物和PDK1分别磷酸化Akt丝氨酸（473位）和苏氨酸（308位），是Akt活化的可靠的预测因子。由Akt磷酸化促凋亡蛋白Bad丝氨酸（112位）和多功能激酶GSK-3β丝氨酸（9位），抑制其活性，促进细胞存活。mTOR是一个重要的信号中心，是两个大分子复合物TORC1和TORC2的一个重要组成部分。 mTOR丝氨酸（2448位）磷酸化，整合生长因子信号和养分供应，在细胞生长和动态平衡中发挥了重要作用。mTORC1磷酸化P70 S6激酶苏氨酸（389位），导致激酶活化和细胞周期的进程。S6核糖体蛋白在p70 S6激酶下游被发现，其丝氨酸（235/236位）的磷酸化反应了mTOR通路的活化作用，并可预测细胞周期进程。AMPK是一个能量传感器，由苏氨酸（172位）磷酸化激活，响应AMP水平升高。 AMPK调节脂肪酸代谢，以及调节蛋白质合成和细胞生长。HSP27是细胞应激的调控者，能够抵抗不良环境的变化。在p38 MAPK通路内，HSP27在丝氨酸（78位）磷酸化。p53在细胞应答DNA损伤和其他基因组畸变中起着重要的作用。通过ATM/ATR 或DNA-PK磷酸化，p53丝氨酸（15位）响应DNA损伤，导致其稳定化并积累。Caspase-3是一个细胞凋亡的关键执行者。 Caspase-3由内蛋白水解分裂天冬氨酸（175位）而被激活，并通过裂解细胞靶点，发挥其促凋亡活性。PARP是一种参与DNA修复的酶，是活化的caspase-3的主要底物之一。天冬氨酸（214位）的裂解导致PARP失活。裂解的caspase-3和裂解的PARP水平增加，是细胞凋亡的可靠指标。

1. Manning, B.D. and Cantley, L.C. (2007) Cell 129, 1261-74.
2. Huang, J. and Manning, B.D. (2009) Biochem Soc Trans 37, 217-22.
3. Dufner, A. and Thomas, G. (1999) Exp Cell Res 253, 100-9.
4. Hardie, D.G. et al. (2012) Nat Rev Mol Cell Biol 13, 251-62.
5. Rubinfeld, H. and Seger, R. (2005) Mol Biotechnol 31, 151-74.
6. Keshet, Y. and Seger, R. (2010) Methods Mol Biol 661, 3-38.
7. Cuadrado, A. and Nebreda, A.R. (2010) Biochem J 429, 403-17.
8. Brognard, J. and Hunter, T. (2011) Curr Opin Genet Dev 21, 4-11.
9. Hunter, T. (2009) Curr Opin Cell Biol 21, 140-6.
10. Manning, G. et al. (2002) Science 298, 1912-34.
11. Kurokawa, M. and Kornbluth, S. (2009) Cell 138, 838-54.
12. Shi, Y. (2004) Cell 117, 855-8.
13. Boatright, K.M. and Salvesen, G.S. (2003) Curr Opin Cell Biol 15, 725-31.

原厂资料：

Specificity / Sensitivity

PathScan® Intracellular Signaling Array Kit (Fluorescent Readout) detects the indicated cellular proteins and signaling nodes only when phosphorylated or cleaved at the specified residues. (see Array Target Map). No significant cross-reactivity has been observed between targets. This kit is optimized for cell lysates diluted to a total protein concentration between 0.2 and 1 mg/ml (see kit protocol). All capture antibodies have been validated for human and mouse-derived samples.

Description

The PathScan® Intracellular Signaling Array Kit (Fluorescent Readout) is a slide-based antibody array founded upon the sandwich immunoassay principle. The array kit allows for the simultaneous detection of 18 important and well-characterized signaling molecules when phosphorylated or cleaved. Target-specific capture antibodies have been spotted in duplicate onto nitrocellulose-coated glass slides. Each kit contains two 16-pad slides, allowing the user to test up to 32 samples and generate 576 data points in a single experiment. Cell lysate is incubated on the slide followed by a biotinylated detection antibody cocktail. Streptavidin-conjugated DyLight® 680 is then used to visualize the bound detection antibody. A fluorescent image of the slide can then be captured with a digital imaging system and spot intensities quantified using array analysis software.

Background

Phosphorylation and proteolysis are two widespread covalent post-translational modifications that represent important regulatory mechanisms in biology. Detection of these modifications on a set of cellular proteins playing a well-understood role in cell biology can provide a broad snapshot of intracellular signaling. 
 The MAPK/Erk cascade is one of the best characterized and widely studied signaling modules. It is involved in a broad range of cellular processes such as proliferation, differentiation, and motility. MAPK/Erk is activated by a wide range of extracellular signals including growth factors, cytokines, hormones, and neurotransmitters. It is activated by dual phosphorylation at Thr202 and Tyr204 by the dual specificity kinases MEK1 and MEK2. 
 p38 and JNK MAPKs are core components of two additional structurally related signal transduction modules. p38 and JNK are activated through a similar dual phosphorylation mechanism by various MAPK kinases in response to pro-inflammatory cytokines, stressful conditions, or genotoxicity. 
 Stat1 and Stat3 are important signaling molecules that are involved in immunity and inflammation and can be activated by a variety of cytokines or growth factors. Stat1 and Stat3 are phosphorylated at Tyr701 or Tyr705, respectively, by cytokine receptor-tethered tyrosine kinases of the Jak family or, in some cases, by other tyrosine kinases such as Src. 
 Akt is a protein kinase generally activated in response to growth factor stimulation that transmits growth and survival signals. Phosphorylation of Akt at Ser473 and Thr308 by TORC2 complex and PDK1, respectively, are reliable predictors of Akt activation. Phosphorylation of PRAS40 at Thr246 by Akt relieves PRAS40 inhibition of TORC1. Akt phosphorylation of the pro-apoptotic protein Bad at Ser112 and the multifunctional kinase GSK-3β at Ser9 inhibits their activity and promotes cell survival. 
 mTOR is an important signaling hub that is a major component of two macromolecular complexes, TORC1 and TORC2. mTOR is phosphorylated at Ser2448 and integrates growth factor signaling and nutrient availability, thus playing an important role in cell growth and homeostasis. mTORC1 phosphorylates p70 S6 Kinase at Thr389, leading to kinase activation and cell cycle progression. The S6 ribosomal protein is found downstream of p70 S6 Kinase and its phoshporylation at Ser235/236 reflects mTOR pathway activation and predicts cell cycle progression. 
 AMPK is an energy sensor that is activated by phosphorylation at Thr172 in response to elevated AMP levels. AMPK regulates fatty acid metabolism, as well as modulates protein synthesis and cell growth. 
 HSP27 is a mediator of cell stress that confers resistance to adverse environmental change. HSP27 is phosphorylated at Ser78 within the p38 MAPK pathway. 
 p53 plays an important role in cellular response to DNA damage and other genomic aberrations. Phosphorylation of p53 at Ser15 by ATM/ATR or DNA-PK in response to DNA damage leads to its stabilization and accumulation. 
 Caspase-3 is a critical executor of apoptosis. Caspase-3 is activated by endoproteolytic cleavage at Asp175 and exerts its pro-apoptotic activity through cleavage of multiple cellular targets. PARP, an enzyme that is involved in DNA repair, is one of the main substrates of activated caspase-3. Cleavage at Asp214 leads to PARP inactivation. Increased levels of cleaved caspase-3 and cleaved PARP are reliable indicators of apoptosis.

1. Manning, B.D. and Cantley, L.C. (2007) Cell 129, 1261-74.
2. Huang, J. and Manning, B.D. (2009) Biochem Soc Trans 37, 217-22.
3. Dufner, A. and Thomas, G. (1999) Exp Cell Res 253, 100-9.
4. Hardie, D.G. et al. (2012) Nat Rev Mol Cell Biol 13, 251-62.
5. Rubinfeld, H. and Seger, R. (2005) Mol Biotechnol 31, 151-74.
6. Keshet, Y. and Seger, R. (2010) Methods Mol Biol 661, 3-38.
7. Cuadrado, A. and Nebreda, A.R. (2010) Biochem J 429, 403-17.
8. Brognard, J. and Hunter, T. (2011) Curr Opin Genet Dev 21, 4-11.
9. Hunter, T. (2009) Curr Opin Cell Biol 21, 140-6.
10. Manning, G. et al. (2002) Science 298, 1912-34.
11. Kurokawa, M. and Kornbluth, S. (2009) Cell 138, 838-54.
12. Shi, Y. (2004) Cell 117, 855-8.
13. Boatright, K.M. and Salvesen, G.S. (2003) Curr Opin Cell Biol 15, 725-31.

注意事项：

*Kit should be stored at 4°C with the exception of 1X Cell Lysis Buffer, which is stored at –20°C (packaged separately).