Assay Method Information

Assay Name:  DNA Gyrase Supercoiling Inhibition Assay
Description:  DNA gyrase supercoiling assays were carried out by mixing the compounds and the enzyme in a similar manner as above (EcTopI relaxation inhibition assay) but in a gyrase assay buffer (35 mM Tris-HCl, 24 mM KCl, 4 mM MgCl2, 2 mM DTT, 1.75 mM ATP, 5 mM spermidine, 0.1 mg/mL BSA, 6.5% glycerol at pH 7.5), followed by the addition of 300 ng of relaxed covalently closed circular DNA (New England Biolabs, Ipswich, Mass., USA) to a final reaction volume of 20 μL. The samples were incubated at 37° C. for 30 minutes before being terminated by the addition of a buffer containing 5% SDS, 0.25% bromophenol blue, and 25% glycerol. The reactions were then analyzed by agarose gel electrophoresis.
Affinity data for this assay
 

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