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Topo IV (from Escherichia coli) is prepared by overexpressing the parC and parE subunits in E. coli and purifying them by methods adapted from Peng and Marians, 19991.
The parC and parE subunits are purified to >95% purity as judged by SDS-PAGE. The topo IV is supplied as a heterotetramer complex in Dilution buffer.
It is recommended that the enzyme is aliquoted to avoid repeated freeze-thaw cycles. Store at -80ºC.
All enzyme is supplied with 5X concentrated Assay Buffer and Dilution buffers which are also available separately.
See technical documents below for more detailed information and lot specific activities.
These contain the enzyme and the supercoiled DNA substrate in addition to the Assay and Dilution buffers for relaxation reactions. 1 U of topo IV will relax 0.5 µg supercoiled pBR322 DNA in 30 minutes at 37°C.
These contain the enzyme and the catenated kDNA substrate in addition to the Assay and Dilution buffers for decatenation reactions. 1 U of topo IV will decatenate 200 ng of kDNA when incubated in 1X Assay buffer in a total reaction volume of 30 µl at 37°C for 30 minutes.
These kits are designed for assaying cell extracts and partially purified fractions containing over-expressed topo IV and contain supercoiled DNA substrate, Assay buffer, Dilution buffer, control relaxed DNA and stop buffer/loading dye.
These kits are designed specifically for cleavage reactions. They contain topo IV enzyme, supercoiled pBR322 DNA substrate and the Assay and Dilution buffers required for DNA cleavage reactions in addition to the enzyme and linearised pBR322 marker.
Cleavage specific enzyme available separately on request.
These kits can be used to test the effects of potential ATPase inhibitors. For example, the coumarin drugs such as novobiocin inhibit the action of topoisomerase IV by competitively inhibiting the hydrolysis of ATP thus preventing supercoiling.
These assays are microtitre plate-based and thus large numbers of compounds can be screened in a relatively short period of time. They also continuous assays which can provide more information than an end point assay.
The kit is supplied with sufficient E. coli topo IV enzyme, plasmid DNA substrate, buffers and other assay components* for 100 assays. The enzyme is supplied at a concentration of 10 U/μl in Dilution Buffer. The kit is also supplied with sufficient wash buffers for one 96-well plate. These buffers are supplied as 20X concentrates and must be diluted with ultra pure water prior to use.
More information about this assay can be found on the 'Services' page under 'High/Medium Throughput Assay'.
Kit issued with limited licence for individual use only.
Patent held by Inspiralis Ltd., Norwich, Norfolk, UK. (Patent No. GB0424953.8, US7838230)