商品编号


TG1009-1



品牌


TopoGEN



公司


TopoGEN



公司分类


Drug screening kits



Size

100 Assays

商品信息




Topoisomerase II Drug Screening Kit Product Description

Topo II inhibitors have been shown to possess effective antibacterial, antiviral and anticancer activity. Identification of topo II inhibitors and antago
NIST
s requires tractable biochemical assays employing purified enzyme.? The Topoisomerase II Drug Screening Kit contains the reagents necessary to screen for agents that affect enzymatic activity.? To allow the customer maximum flexibility, purified topoisomerase II may be purchased separately from the kit (TG2000H).? This kit will allow detection of two classes of topoisomerase inhibitors: those that stimulate formation of cleavable complexes and those that antagonize topo II action on the DNA.? Also included in this kit is detailed documentation on design and interpretation of inhibition data.? Known inhibitors are included as controls.? The DNA substrate (pHOT1) included in this assay is ideal for these studies because it contains a single, high affinity topo II cleavage site (note that topo II is not particularly sequence specific). The assay system is based upon evaluating the formation of DNA cleavage products which may be nicked, open circular DNA or linear DNA. The products are then resolved by ethidium bromide gel electrophoresis as described in the kit.


Topoisomerase II Drug Screening Kit Contents (representative of 100 assay kit size):

-Supercoiled DNA [pHOT1 DNA] 25 ug
-
Marker
s are linear pHOT1 DNA and open circular DNA
-10X Topoisomerase II assay buffer components*


*To improve st
ABI
lity, the 10x Topo II Assay buffer is provided as two components:?Buffer A (no ATP) and Buffer B (ATP). The Complete 10x Assay buffer is made fresh each time by combining Buffer A and B.

-Sodium dodecyl sulfate (10%)
-10X Gel lo
ADI
ng buffer (bromophenol blue, glycerol)
-Control inhibitor, Etoposide
-Proteinase K
-Detailed instruction manual

Note: Topo II enzyme is sold separately.


Topo II Drug Screening Background:

This is a cleavage based detection kit that will reveal whether a particular agent either antagonizes the action of topo II or affects the cleavage/religation cycle.? Reactions are assembled in microcentrifuge tubes using reagents supplied in the kit (buffer, DNA, control inhibitor).? This kit is designed to work with purified topoisomerase II provided by
TopoGEN
.? After adding enzyme, the reactions are incubated 15-30 min (37°C), and terminated by addition of SDS.


A typical reaction:


H2O: Volume variable, make up to volume (20 ul in this case)

10X Assay buffer 2 ?l (made fresh from Buffers A and B, see Kit details)

DNA (supercoiled) 1 ?l

Test Compound or control inhibitor: Variable (limited by solvent effects as addressed in controls)

**Purified Topoisomerase II: 4-6 Units (enzyme sold separately)

Incubate 15-30 minutes at 37°C

Add 2 ?l SDS; digest with proteinase K (50 ug/ml)

Add 0.1 vol sample lo
ADI
ng dye (supplied)

Load gel: 1% agarose containing 0.5 ug/ml ethidium bromide along with the
Marker
DNAs included in the kit.




The reactions indicated above each lane in the figure were carried out using the enzyme provided by
TopoGEN
and 1 ul of DNA (0.25 ug of form I pHOT-1, as provided in each kit).? The reaction products were analyzed on a 1% agarose gel; the running buffer contained 0.5 ug/ml ethidium bromide.? The gel was then destained in distilled water prior to photodocumentation.? Formation of the cleavable complex (linear DNA species) is clearly vis
IBL
e.? Additionally, relaxation of supercoiled DNA substrate (form I pRYG DNA) was inhibited in all reactions that contained VM26.? The kit contains
Marker
DNAs, VM-26 control inhibitor, all buffers and complete instructions on using the reagents to screen for inhibitors of topoisomerase II.? The manual specifies conditions for gel electrophoresis to resolve all DNA forms; publication quality gels will result.

**Cleavage reactions require more topo II than relaxation assays since the former is a stoichiometric reaction of enzyme.? For example, the above reactions contained 4 units of enzyme (units defined by decatenation activity).


Reviews and Citations:


Sudan, S., & Rupasinghe, H. V. (2014).
Flavonoid-Enriched Apple Fraction AF4 Induces Cell Cycle Arrest, DNA Topoisomerase II Inhibition, and Apoptosis in Human Liver Cancer HepG2 Cells.

Nutrition and cancer
, ahead-of-print (2014): 1-10.? doi
:
10.1080/01635581.2014.951733

Cheng MH, Yang YC, Wong YH, Chen TR, Lee CY, Yang CC, Chen SH, Yang IN, Yang YS, Huang HS, Yang CY, Huang MS, Chiu HF:
B1, a novel topoisomerase II inhibitor, induces apoptosis and cell cycle G1 arrest in lung adenocarcinoma A549 cells.
?
Anti-Cancer Drugs
2012, 23(2): 191-199.? doi: 10.1097/CAD.0b013e32834cd277
?

Shi W, Marcus SL, Lowary TL:
Cytotoxicity and topoisomerase I/II inhibition of glycosylated 2-phenyl-indoles, 2-phenyl-benzo[b]thiophenes and 2-phenyl-benzy[b]furans
.?
Bioorganic and Medicinal Chemistry
2010.? doi: 10.1016/j.bmc.2010.10.054

Gong Y, Firestone GL, Bjeldanes LF:
3,3'-Diindolylmethane is a novel topoisomerase IIa catalytic inhibitor that induces S-phase retardation and mitotic delay in human hepatoma HepG2 cells.
?
Molecular Pharmacology
2006, 69(4): 1320-1327.