• 产品编号:DNACasp3
    • 产品名称:Fluoro ssDNA Caspase 3™ - DNACaspTM
    • 产品规格:25Test/50Test
    • 价  格:4875/9375
    • 供 应 商:Cell Tech
    • 库  存:
  • Fluoro ssDNA Caspase 3™ - DNACaspTM
    A Dual antibody assay to simultaneously detect DNA Damage (Single Stranded) & active Caspase 3 in apoptotic cells
    Key Benefits:
    • Much more Reliable as compared to TUNEL assays - No False positive signals
    • Multiple parameter detection of DNA damage and caspase 3 activity simultaneously
    • Readout - Flow cytometry, Fluorescence 96 well plate reader, Fluorescence microscope
    • Yields both quantitative and qualitative results. Gives a strong positive signal.
    • Ease Of Use: Dual parameter results to confirm apoptosis in cells.
    • Compatible with human, mouse, rat, bovine, porcine species
    Introduction:
    1. Antibody to Single Stranded DNA
    A widely used cytochemical technique for evaluation of DNA damage associated with apoptosis is the terminal deoxynucleotidyl transferase-mediated in situ end labeling or TUNEL assay. However the TUNEL assay has its drawbacks in that false positive staining makes the assay unreliable as a marker for apoptosis 1-5. A more universal and specific marker for detecting apoptosis associated DNA damage is to measure the morphological changes in nuclei that reflect chromatin condensation into compact masses. 6-7. Further biochemical and cytochemical studies have demonstrated the increased susceptibility of apoptotic DNA to thermal denaturation. Analysis of nuclei by scanning calorimetry to detect thermal induced DNA denaturation and analysis of DNA fragmentation by electrophoresis have shown that intact apoptotic DNA is susceptible to denaturation at lower temperatures then that of non-apoptotic cells 8.
      2. Antibody to Active Caspase 3
    Apoptosis is an evolutionarily conserved form of cell suicide, which follows a specialized cellular process. The central component of this process is a cascade of proteolytic enzymes called caspases. These enzymes participate in a series of reactions that are triggered in response to pro-apoptotic signals and result in cleavage of protein substrates, causing the disassembly of the cell 13. Caspases have been identified in organisms ranging from C. elegans to humans. The mammalian caspases play distinct roles in apoptosis and inflammation. In apoptosis, caspases are responsible for proteolytic cleavages that lead to cell disassembly (effector caspases), and are involved in upstream regulatory events (initiator caspases). An active caspase consists of two large (~20 kD) and two small (~10 kD) subunits to form two heterodimers which associate in a tetramer 14-16. As is common with other proteases, caspases are synthesized as precursors that undergo proteolytic maturation, either autocatalytically or in a cascade by enzymes with similar specificity 17. Caspase 3, also known as CPP-32, Apopain or Yama, is a key effector caspase in the apoptotic pathway 18. It is present in many different cell lineages and is responsible for the cleavage of a variety of molecules such as poly ADP-ribose polymerase (PARP), protein kinase Cd, actin and DNA-dependent protein kinase 19-20.
    Assay Principle:
    Cell Technology introduces a dual parameter antibody based assay to detect DNA damage (heat denatured single stranded DNA: ssDNA)9-12 and active caspase 3 in apoptotic cells. The assay utilizes a monoclonal antibody generated against ssDNA and a primary rabbit affinity purified polyclonal antibody raised against amino acid 163-175 of murine caspase 3 12. This neo epitope is present on the p18 subunit of cleaved caspase 3 21.
    DNA Damage detection: Excitation: 488nm, Emission: 530nm (FL1)
    Caspase 3 detection: Excitation: 633nm, Emission: 670nm (LP) (FL4)
    References
    1. Charriaut–Marlangue C, Ben-Ari J (1995) A cautionary note on the use of TUNEL to determine apoptosis. NeuroReport 7:61–64
    2.Grasl–Kraipp B, Ruttkau–Nedecky B, Koudelka H, Bukowska K, Bursch W, Schulte–Hermann R (1995) In situ detection of frag-mented DNA (TUNEL) fails to discriminate among apoptosis, necrosis and autolytic cell death: a cautionary note. Hepatology 21:1465–146
    3. Didenko VV, Hornsby PJ (1996) Presence of double-stranded DNA breaks with single-base 39 overhangs in cells undergoing apopto-sis but not necrosis. J Cell Biol 135:1369–1376
    4. Ohno M, Takemura G, Ohno A, Misao J, Hayakawa Y, Minatogu-chi S, Fujiwara T, Fujiwara H (1998) “Apoptotic” myocytes in infarct area in rabbit hearts may be oncotic myocytes with DNA fragmentation: analysis by immunogold electron microscopy combined with in situ nick end-labeling. Circulation 98:1422–1430.
    5. Stadelmann C, Bruck W, Bancher C, Jellinger K, Lassmann H (1998) Alzheimer disease: DNA fragmentation indicates in-creased neuronal vulnerability, but not apoptosis. Neuropathol Exp Neurol 57:456–464
    6. Willingham MC (1999) Cytochemical methods for the detection of apoptosis. J Histochem Cytochem 47:1101–1109
    7. Zamzani N, Kroemer J (1999) Condensed matter in cell death. Nature 401:127–128.
    8. Allera C, Lazzarini G, Patrone E, Alberti I, Barboro P, Sanna P, Melchiori, A, Parodi S, Balbi C (1997) Condensation of chromatin in apoptotic thymocytes shows a specific structural change. J. Biol Chem 272:10817–10822.
    9. Frankfurt OS (1990) Decreased DNA stability in cells treated with alkylating agents. Exp Cell Res 191:181–185.
    10. Frankfurt OS, Robb JA, Sugarbaker EV, Villa L (1996) Monoclonal antibody to single-stranded DNA is a specific and sensitive cellular marker of apoptosis. Exp Cell Res 226:387–397.
    11. Frankfurt OS (1994) Detection of apoptosis in leukemic and breast cancer cells with monoclonal antibody to single-stranded DNA. Anticancer Res 14:1861–1870.
    12. Oskar S. Frankfurt and Awtar Krishan (2001). Identification of Apoptotic Cells by Formamide-induced DNA Denaturation in Condensed Chromatin. The Journal of Histochemistry & Cytochemistry Volume 49(3): 369–378, 2001
    13. Slee, E. A., C. Adrain, and S. J. Martin. 1999. Serial Killers: ordering caspase activation events in apoptosis. Cell Death and Differ. 6:1067-1074.
    14. Walker, N. P., R. V. Talanian, K. D. Brady, L. C. Dang, N. J. Bump, C. R. Ferenz, S. Franklin, T. Ghayur, M. C. Hackett and L. D. Hammill. 1994. Crystal Structure of the Cysteine Protease Interleukin-1ß-Converting Enzyme: A (p20/p10)2 Homodimer. Cell 78:343-352.
    15. Wilson, K. P., J. F. Black, J. A. Thomson, E. E. Kim, J. P. Griffith, M. A. Navia, M. A. Murcko, S. P. Chambers, R. A. Aldape, S. A. Raybuck, and D. J. Livingston. 1994. Structure and mechanism of interleukin-1 beta converting enzyme. Nature 370: 270-275.
    16. Rotonda, J., D. W. Nicholson, K. M. Fazil, M. Gallant, Y. Gareau, M. Labelle, E. P. Peterson, D. M. Rasper, R. Ruel, J. P. Vaillancourt, N. A. Thornberry and J. W. Becker. 1996. The three-dimensional structure of apopain/CPP32, a key mediator of apoptosis. Nature Struct. Biol. 3(7): 619-625.
      17. Kumar, S. 1999. Mechanisms mediating caspase activation in cell death. Cell Death and Differ. 6: 1060-1066.
      18. Alnemri, E.S. et al (1996) Cell 87:171
      19. Trends Biochem Sci 22,388 (1997)
      20. Nature 376, 37 (1995).
      21. Srinivasan A, et al Cell Death and Differentiation (1998) 5: 1004-1016
    Kit contents and Long Term storage:
    1. Mouse anti single stranded DNA…………………  Part # 1003              -70°C aliquot
    2. Rabbit anti active Caspase 3 …………………  Part # 1006                   4-8°C
    3. Goat Anti mouse FITC labeled………… Part # 2006                           4-8°C
    4. Goat Anti Rabbit APC ………… Part # 2007                                       4-8°C
    5.  Fixative…………………………..…………………………… Part # 3033                    -20°C

    6. 10 X Wash Buffer……………………………………………    Part # 3037               -20°C

    7. 1X Denaturing Buffer ……………………………Part # 3036           Room Temp
    The following kits are available:
    Catalog #
    Size* Price
    DNACasp3-1 
    25 Tests 4875.00
    DNACasp3-2  50 Tests 9375.00 

    Fluoro ssDNA Caspase 3™ - DNACaspTM

    一对抗体,同时检测凋亡细胞中DNA损伤(单链)及caspase 3 的活性

     

    主要优点:

    TUNEL法相比,该方法检测结果更可靠---无假阳性信号。

    多参数同时检测DNA损伤及caspase 3的活性

    读数 - 流式细胞仪,荧光96孔酶标仪,荧光显微镜。

    定量和定性结果。有强烈的阳性信号。

    易用性:双参数结果证实细胞凋亡。

    适合于人类,小鼠,大鼠,牛,猪。

     

    介绍:

    1. 单链DNA抗体

    广泛应用的细胞化学技术用于评估与凋亡相关的DNA损伤的方法是末端脱氧核苷酸转移酶介导的原位末端标记法或者TUNEL法。然而,TUNEL法有假阳性染色的缺点,使得细胞凋亡检测结果不可靠1-5。一种检测细胞凋亡相关联的DNA损伤的更具有普遍性和特异性的标志是检测细胞核形态学变化,从而反映染色质凝聚成紧密的物质形态6-7。进一步的生化和细胞化学的研究已证实了凋亡的DNA的热变性。用扫描量热法做核心分析来检测热引起的DNA变性和电泳分析DNA片段,表明在较低温度下完整的凋亡DNA比非凋亡的DNA容易变性8

    2. 活性Caspase 3抗体

    细胞凋亡是一种细胞程序性死亡的过程,它遵循一个专门的细胞进化过程。这个过程的核心组件是一系列蛋白水解酶称为caspases。这些酶参与一系列的反应,触发凋亡信号和蛋白质底物的裂解,从而使细胞分解(13)。半胱氨酸蛋白酶存在于从线虫到人类的生物体中。哺乳动物的半胱氨酸蛋白酶在细胞凋亡和炎症中有独特作用。在细胞凋亡中,半胱氨酸蛋白酶的水解作用造成细胞解体(效应半胱氨酸蛋白酶),并参与上游调控(启动半胱氨酸蛋白酶)。一种活性半胱氨酸蛋白酶由两个大亚基(20kDa)和两个小亚基(10kDa)组成,形成两个异质二聚体,并与四聚体(14-16)联系。由于与其他蛋白酶相同,合成的半胱氨酸蛋白酶进行蛋白前体的成熟,无论是自动催化或具有类似的特异性的酶的级联(17)。caspase 3也叫做cpp - 32ApopainYama,是细胞凋亡途径中的一种重要效应半胱氨酸蛋白酶 18。它存在于许多不同的细胞系中,负责各种分子如多聚ADP -核糖聚合酶(PARP),蛋白激酶Cd,肌动蛋白和DNA依赖的蛋白激酶的裂解19-20

    检测原理:

    Cell Technology介绍了一种基于双参数抗体检测凋亡细胞中DNA损伤(热变性的单链DNAssDNA9-12及活性caspase 3的方法。该法采用了单链DNA单克隆抗体和亲和纯化的兔抗鼠caspase 3163-175氨基酸片段的多克隆抗体12。这一新的抗原表位是在裂解的caspase 3 p18亚基上21

    DNA损伤检测:激发波长:488nm,发射波长:为530nmFL1

    caspase 3的检测:激发波长:633nm波长,发射波长:670nmLP)(FL4