商品编号


TG1018-1



品牌


TopoGEN



公司


TopoGEN



公司分类


Drug screening kits



Size

100 Assays

商品信息


Topoisomerase I Drug Screening Kit Product Description
The Topo I Drug Kit introduces transient nicks in DNA at specific sites.? Detection of these transient DNA nicks requires trapping the enzyme on DNA in a nicked intermediate complex using protein denaturants. The resulting covalent DNA/topo I complexes contain nicked open circular DNA which can be detected by agarose gel electrophoresis (with ethidium bromide).? Trapping nicked intermediates is relatively inefficient; however, inhibitors, such as the natural product camptothecin, st
ABI
lize the intermediate and lead to an increase in the nicked DNA product.? This forms the basis for a mecha
NIST
ic drug screening assay designed to allow detection of agents that affect

topoisomerase I by st
ABI
lizing the cleaved intermediate complex.? The
TopoGEN
Topo I Drug Screening Kit is designed to allow the investigator to quickly identify novel inhibitors of topo I enzyme (sold separately).? The kit will allow detection of novel compounds that either st
ABI
lize the nicked intermediate or otherwise inhibit catalytic activity of topoisomerase I.


Topoisomerase I Drug Screening Kit Contents (representative for 100 assay kit size):

-Supercoiled pHOT-1 DNA, 25 ?g
-Relaxed and nicked plasmid DNA
Marker
s
-10X Topoisomerase I assay/cleavage buffer, 300 ul
-Sodium dodecyl sulfate (SDS) termination buffer (10%)
-10X gel lo
ADI
ng dye
-Control inhibitor (Camptothecin; lyophilized)
-Proteinase K
-Detailed instruction manual


Note:
Topoisomerase I enzyme sold separately.

The Topoisomerase I Drug Screening Kit is designed to work with human topo I available from
TopoGEN
. Since cleavage assays require more enzyme than catalytic assays,
TopoGEN
sells the enzyme in larger lots which takes this difference into account.



Use of the Topoisomerase I Drug Kit
Reactions indicated above each lane were carried out using 5 units of
TopoGEN
type I topoisomerase.? The reactions were incubated for 30 min at 37 C and stopped with SDS.? After digestion with proteinase K (50 ug/ml, 60 min at 37 C), samples were loaded onto a 1% agarose gel.? Under these conditions, with excess topo I and camptothecin, significant conversion of supercoiled (form I) DNA to open circular (OC DNA) was observed.? It is important to recognize that cleavage assays will consume more enzyme than catalytic (relaxation type) assays.? This is because each cleavage event consumes one enzyme molecule; i.e., the reaction requires stoichiometic amounts of topo I.