E. coli Topoisomerase IV Drug Screening Kit

The Topoisomerase IV Drug Screening Kit contains reagents necessary to identify compounds that affect enzyme activity.  Note that kits with a catalog number denoted -A will include E. coli topoisomerase IV enyzme.  This kit will allow detection of two kinds of topoisomerase inhibitors: those that stimulate formation of cleavable complexes and those that antagonize topoisomerase (topo) IV action on the DNA.  Since topo IV is a type II enzyme, we may refer to it as either topo IV or topo II.  A known topo II poison is included as a control.  The assay system is based upon incubating supercoiled plasmid (pHOT1) with Topo IV and 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 IV Drug Screening Kit Contents (suitable for 100 assays):

-100 Units Topoisomerase IV Enzyme (available with catalog no. TG1007-1A)
-pHOT1 DNA, supercoiled 25 ug
-Marker DNA, Linear pHOT1
-Incomplete Buffer A contains the following: 200 mM HEPES-KOH [pH7.6], 500 mM potassium glutamate, 50 mM Magnesium Acetate, 50 mM Dithiothreitol, 250 ug BSA/ml (500 ul)*
-ATP Buffer B contains 100 mM ATP*

*These two buffers must be mixed before use to make the final 5x Complete Topo IV Assay Buffer (A+B) as follows: add 0.1 volume of Buffer B to 1.0 volume Buffer A. Prepare only as much of the Complete Topo IV Assay Buffer (A+B) as needed for each day or set of experiments. Note that this buffer should be kept on ice and used the same day, then discarded.

-Ciprofloxacin control drug
-10% sodium dodecyl sulfate (SDS)
-10x gel loading buffer
-Proteinase K
-Detailed instruction manual

The kit may be shipped at ambient temperature or on ice (dry ice or wet ice).  The DNAs should be stored at 4°C and the buffers stored at -20°C upon receipt. Avoid frequent freeze/thaw cycles with the plasmid as this may contribute to DNA breakage.  Store Enzyme at -70° C.

Topoisomerase-IV-Drug-Kit-Protocol

Reviews and Citations

  • Phillips JW, Goetz MA, Smith SK, Zink DL, Polishook J, Onishi R, Salowe S, Wiltsie J, Allocco J, Sigmund J, Dorso K, Lee S, Skwish S, de la Cruz M, Martin J, Vicente F, Genilloud O, Lu J, Painter RE, Young K, Overbye K, Donald RGK, Singh SB: Discovery of Kibdelomycin, A Potent New Class of Bacterial Type II Topoisomerase Inhibitor by Chemical-Genetic Profiling in Staphylococcus aureus.  Cell Chemistry and Biology 2011, 18: 955-965.  doi: 10.1016/j.chembiol.2011.06.011.

 

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