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Name
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Topoisomerase II In Vivo Link Kit 200 Assays
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Price
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$469.00
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SKU/Part Number
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1022
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Extended Description
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TopoGEN has extensive experience with assays for topoisomerase inhibition in vivo. These experiments allow the investigator to ascertain whether a novel agent is active against endogenous topo in a chromosomal setting in nuclei. An important benefit to this analysis is that one can use any tumor cell or tissue in order to establish clinical efficacy of a test drug against a specific tumor cell line. We use essentially the same basic approach for Topo I as for Topo II. The methods are based upon physically separating the topo/DNA adducts from free DNA and using antibodies to measure bound topo I or II. In this analysis, tissue culture cells are treated with a test compound along with negative controls (no drug) and positive controls (with known inhibitors). The cells are drug treated and rapidly lysed with Sarkosyl which traps some fraction of the endogenous topo on DNA in a covalent cleavage complex. Following detergent lysis, the lysate is diluted to fully dissociate non-covalent DNA/protein complexes. Covalent topo/DNA complexes are resolved on a step CsCl gradient (ionic DNA/protein interactions are also prevented by 5 M CsCl). The gradient resolves DNA, chromatin aggregates, and protein, respectively. Gradients are centrifuged overnight and fractionated. The amount of topo coincident with the DNA peak is a measure of covalent DNA/topo complexes. Topo concentration in the DNA peak is determined by imunoblotting using antibody to topo I or II as probe. In the absence of agents that stabilize the cleavable complex (etoposide, camptothecin) only low levels of topo are found in the DNA peak; this is particularly obvious with topo II since the type II enzyme is not trapped by this method unless an inhibitor is used. In contrast, topo I is trapped to a low extent even in the absence of camptothecin. The ratio of topo at the DNA density and the protein density reflects the relative efficiency of stabilization of the cleavable complexes.
References:
Ebert et al., J. Virology 64: 4069-4066, 1990.
Muller and Mehta, Mol. Cell. Biol. 8: 3661-3669, 1988.
Trask and Muller, Proc. Natl. Acad. Sci. 85:1417-1421.
Kit Contents:
-Camptothecin (supplied lyophilized with Topo I Kits Cat# 1021)
-Etoposide (supplied lyophilized with Topo II Kits Cat#1022)
-Sarkosyl (20%)
-Antibody to Topo I (Supplied with Topo I Kits, Cat# 1021)
-Antibody to Topo II (170 kDa form, supplied with Topo II Kits, Cat# 1022)
-CsCl Stock Solution
-Detailed Instruction Manual
Shipping and Recommended Storage Conditions:
This kit is shipped at ambient temperature (-20 C Storage).
Related Products: Rabbit Antibody against Human p170 Topo II (Cat. No. 2011), Scleroderma Antibody against Human Topo I (Cat. No. 2012), Rabbit Antibody against Human Topo I (Cat. No. 2012-2)
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