E. coli Topoisomerase IV

Cat. No.: E418746

Panoramica

Product Info

Included Materials:

A 2-part assay buffer and dilution buffer are included.

Description

Topo IV is a type II topoisomerase that is encoded by two genes parC and parE in E. coli.  It is essential for decatenation and chromosomal segregation in E. coli; thus, topo IV tends to prefer intermolecular strand passing (catenating and decatenating reactions) over relaxation reactions.  Topoisomerase IV is also the target for quinolone-based antibacterial agents which act by subverting the enzyme into a DNA damaging agent. Aladdin offers purified Topo IV for use in all aspects of drug development and screening assays.  The enzyme is very active in decatenating kDNA (TG2013) but will also relax supercoiled plasmid DNA.  It is less robust in relaxation activity, however.  Topo IV is purified according to Peng and Marians.

Shipping&storage

The enzyme is shipped on dry ice and should be stored at -70°C. We also recommend that the enzyme be aliquoted after the first thaw. Note that repeated rounds of freeze/thaw will lead to activity loss.

Quality Control Tests

Topo IV was overexpressed and purified using the published methods.  A single band on SDS-PAGE was detected by CB staining for each subunit.  Cross contamination by topo I was assessed by assaying for relaxation of supercoiled DNA under conditions optimized for type I activity.  Under these conditions, after 2 hours of incubation with pBR322, no relaxation products were detectable.

Assay Conditions

Reactions contained the following (20 ul): 40 mM Tris-HCl (pH 7.5), 6 mM MgCl2, 10 mM DDT, 100 mM potassium glutamate, 40 uM ATP, 50 ug BSA/ml, 0.2 ug kDNA.  Reactions were terminated after 30 min at 37°C with SDS (1%) followed by proteinase K digestion (50 ug/ml, 15 min at 56°C).  Reaction products were run on a 1% agarose gel containing 0.5 ug/ml ethidium bromide (monitored with hand held UV light to resolve decatenation products), destained with distilled water (20 min room temperature) and photographed.

Specifications

Product Name

E. coli Topoisomerase IV

Tipo di molecola

Piccola molecola

Stoccaggio e spedizione

Condizioni di conservazione di stoccaggio

Store at -80°C,Avoid repeated freezing and thawing

Spedito in

Dry ice packs + Cold packs

Domande frequenti

How should this product be stored?

Store at ?80 °C. Avoid repeated freeze–thaw cycles. Aliquot into single-use volumes before freezing.

How is this product shipped?

This product ships frozen with dry ice and cold packs. Unpack on arrival and transfer it to the storage condition stated above; handle dry ice with insulated gloves in a ventilated area.

What documentation is provided?

Available product documentation, including Certificates of Analysis (COA), Safety Data Sheets (SDS), and specification sheets, is shown in the product document area. Document availability and access follow the current site policy.