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APT1000 | ApopTag® ISOL Dual Fluorescence Apoptosis Detection Kit (DNase Types I & II)

APT1000
25 assays  
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      Overview

      Replacement Information

      Key Specifications Table

      Description
      Catalogue NumberAPT1000
      Trade Name
      • ApopTag
      • Chemicon
      DescriptionApopTag® ISOL Dual Fluorescence Apoptosis Detection Kit (DNase Types I & II)
      OverviewThe ApopTag ISOL Dual Fluorescence Kit utilizes a proprietary double hairpin, dual fluorescently labeled oligonucleotide labeling process to detect and distinguish between typical apoptotic DNA breaks induced by either DNase I or DNase II enzyme activities. The Vaccinia Topoisomerase I mediated ligation reaction is adaptable for staining paraffin-embedded tissue, frozen tissue sections, cell suspensions, and adherent cells. In situ staining for DNA fragmentation (as in the ISOL method) is both a means of detection for rare cells and an analytical test of those cells' DNA. ApopTag ISOL Kits facilitate the differentiation of apoptotic cells from necrotic or transiently damaged cells. While conventional in situ detection techniques such as ISEL (Klenow DNA polymerase), TUNEL (terminal deoxynucleotidyl transferase, TdT) and ISNT (DNA Polymerase I) are useful in detecting internucleosomal DNA cleavage, they do not differentiate DNase Type I and DNase Type II cleavage which results from the activation of apoptotic endonucleases. When ISOL was used for direct comparison with TUNEL in specimens without necrosis, the results have been concordant. In specimens presenting necrosis, the better selectivity of ISOL was proven.
      Background InformationThe ApopTag ISOL Fluorescence Apoptosis Detection (DNase Types I & II) technique is based upon the biochemical specificity of the enzymes T4 DNA ligase and Vaccinia Virus DNA Topoisomerase I and the unique, dual labeled, dual hairpin oligonucleotide (8,9,10). This self-annealing oligo contains two sets of complementary base sequences that spontaneously form two duplex segments resulting in a dual hairpin secondary structure (Figure 1). The oligo also contains two internal fluorescent labels which are located at opposite poles of the dual hairpin structure. At one pole is a FAM internal label and the other pole contains a CR590 label. The basis of the detection mechanism relies on the 5'-CCCTT-3' Topoisomerase I recognition site located in the middle of the dual hairpin structure. The Topoisomerase I cuts the DNA at the 3' end of the recognition site causing the dual hairpin oligo to dissociate into two separate differentially labeled hairpin oligonucleotides. The Toposiomerase I remains covalently bound to the oligo containing the recognition site and the FAM label while the other CR590 containing oligo dissociates. Hence, two differentially labeled hairpin oligo probes are created. The biochemical specificity of the provided enzymes impacts the detection aspect of the protocol, in that Vaccinia Topoisomerase I will recognize and ligate the FAM oligo to 5'-OH 3'-PO4 groups (DNase type II specific cut) whereas T4 DNA Ligase will recognize and ligate the CR590 labeled oligo to 5'-PO4 3'-OH groups (DNase type I specific cut). The ISOL Kit does not label nicks, gaps, single-stranded DNA, 3'-recessed ends or 3'-overhanging ends.
      References
      Product Information
      Components
      • T4 DNA Ligase Enzyme (Part. No.2007460) - 100 μL
      • Dual Reaction Buffer (2X) (Part. No.2007464) - 375 μL
      • Dual Labeled Oligo (Part. No.2007463) - 25 μL
      • Vaccinia Topoisomerase I (Part. No. 2007459) - 250 μL
      • Plastic Coverslips (Part. No. 90421) - 100 each
      • Positive Control Slides (Part. No. 90422) - 2 each
      • Proteinase K (Part. No. 90435) - 25 mg
      Applications
      ApplicationThe ApopTag ISOL Dual Fluorescence Kit utilizes a proprietary double hairpin, dual fluorescently labeled oligonucleotide labeling process to detect & distinguish between typical apoptotic DNA breaks induced by either DNase I or DNase II.
      Biological Information
      Species Reactivity
      • All
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Usage Statement
      • Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
      Storage and Shipping Information
      Storage Conditions1 year at -20°C from date of shipment
      Packaging Information
      Material Size25 assays
      Transport Information
      Supplemental Information
      Specifications

      Documentation

      References | 14 Available | See All References

      Reference overviewPub Med ID
      Acid DNases and their interest among apoptotic endonucleases
      Counis, Marie-France and Torriglia, Alicia
      Biochimie, 88:1851-8 (2006) 2006

      16989934 16989934
      Semi-artificial Fluorescent Molecular Machine for DNA Damage Detection
      Didenko VV, Minchew CL, Shuman S, Baskin DS.
      Didenko, Vladimir V, et al 2004

      17330146 17330146
      Molecular cloning and characterization of human caspase-activated DNase
      Mukae, N, et al
      Proc Natl Acad Sci USA, 95:9123-8 (1998) 1998

      9689044 9689044
      Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis)
      Darzynkiewicz, Z, et al
      Cytometry, 27:1-20 (1997) 1997

      9000580 9000580
      Ultrastructural detection of DNA strand breaks in apoptotic neural cells by in situ end-labelling techniques.
      Migheli, A, et al.
      J. Pathol., 176: 27-35 (1995) 1995

      Show Abstract
      7542332 7542332
      Anatomical methods in cell death
      Kerr, J F, et al
      Methods Cell Biol, 46:1-27 (1995) 1995

      7609651 7609651
      Programmed cell death during mammary gland involution.
      Strange, R, et al.
      Methods Cell Biol., 46: 355-68 (1995) 1995

      Show Abstract
      7609656 7609656
      Microwave irradiation of paraffin-embedded tissue sensitizes the TUNEL method for in situ detection of apoptotic cells
      Sträter, J, et al
      Histochem Cell Biol, 103:157-60 (1995) 1995

      7634155 7634155
      BCR-ABL maintains resistance of chronic myelogenous leukemia cells to apoptotic cell death
      McGahon, A, et al
      Blood, 83:1179-87 (1994) 1994

      8118022 8118022
      Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.
      Gavrieli, Y, et al.
      J. Cell Biol., 119: 493-501 (1992) 1992

      Show Abstract
      1400587 1400587

      Brochure

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      User Guides

      Title
      ApopTag® ISOL Dual Fluorescence Apoptosis Detection Kit (DNase Types I & II)

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      Categories

      Life Science Research > Cell Analysis > Cell-based Assays > Apoptosis / Cell Death Assays