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APT1000

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

The 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.

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UNSPSC Code:
12161503
eCl@ss:
32161000

Key Documents

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species reactivity (predicted by homology)

all

manufacturer/tradename

ApopTag
Chemicon®

shipped in

dry ice

General description

The 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.
The 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.

Application

Research Category
Apoptosis & Cancer

Preparation Note

1 year at -20°C from date of shipment

Other Notes

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

Legal Information

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

Disclaimer

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.

Pictograms

Health hazardExclamation mark
GHS08,GHS07

Signal Word

Danger

Hazard Statements

H315,H319,H334,H335

Hazard Classifications

Eye Irrit. 2 - Resp. Sens. 1 - Skin Irrit. 2 - STOT SE 3

Target Organs

Respiratory system

Storage Class Code

10 - Combustible liquids

Certificates of Analysis (COA)

Search for Certificates of Analysis (COA) by entering the products Lot/Batch Number. Lot and Batch Numbers can be found on a product’s label following the words ‘Lot’ or ‘Batch’.

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Yoshihiro Fukawatase et al.
Scientific reports, 4, 5421-5421 (2014-06-28)
Ataxia telangiectasia is a neurodegenerative inherited disease with chromosomal instability and hypersensitivity to ionizing radiation. iPS cells lacking ATM (AT-iPS cells) exhibited hypersensitivity to X-ray irradiation, one of the characteristics of the disease. While parental ataxia telangiectasia cells exhibited significant
Spermatogonial apoptosis has three morphologically recognizable phases and shows no circadian rhythm during normal spermatogenesis in the rat.
Allan, D J, et al.
Cell proliferation, 25, 241-250 (1992)
BCR-ABL maintains resistance of chronic myelogenous leukemia cells to apoptotic cell death
McGahon, A, et al
Blood, 83, 1179-1187 (1994)
Marie-France Counis et al.
Biochimie, 88(12), 1851-1858 (2006-09-23)
Apoptosis is characterized by cell shrinkage, nuclear condensation and internucleosomal DNA cleavage. Besides the central role of caspases and other proteases, cell death triggers DNA degradation so that DNases have an active role in apoptotic cell death. The best-characterized apoptotic
J Sträter et al.
Histochemistry and cell biology, 103(2), 157-160 (1995-02-01)
Apoptosis is a morphologically distinct form of programmed cell death that plays an important role in the growth regulation of a variety of tissues and also in the elimination of self-reacting immunocompetent cells. Several techniques for the qualitative and quantitative
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