A typical reaction is as follows:
1 U of topo IV will decatenate 200 ng of kDNA when incubated in 40 mM HEPES.KOH (pH 7.6), 100 mM Potassium Glutamate, 10 mM Magnesium Acetate, 10 mM DTT, 1 mM ATP and 50 µg/ml albumin, in a total reaction volume of 30 µl at 37°C for 30 minutes.
The reaction is stopped by the addition of 30 µl chloroform/iso-amyl alcohol and 30 µl of 2X Stop Buffer (STEB), vortexed and centrifuged briefly (5-10 seconds each) before being loaded on an agarose gel.
Gels can be run either in TBE (90 mM Tris.Borate, 2 mM EDTA) or TAE (40 mM Tris.acetate, 2 mM EDTA)
Gels can be run in the presence or absence of ethidium bromide (EtBr) or chloroquine (CQ) which will resolve nicked OC DNA from relaxed (see below).
The substrate for decatenation assays is kDNA which consists of a complex interlinked network of catenated DNA maxi- and mini-circles.
Due to their high molecular mass, these complexes cannot enter an agarose gel under normal electrophesis conditions, but remain in the wells. In the presence of a type II topoisomerase (in this case topo IV) the mini-circles (2.5 Kb) are released by decatenation and can be resolved quickly and easily at relatively high voltages. Decatenation, rather than relaxation, is the preferred reaction for topo IV and human topo II making this an ideal assay for the quick identification of potential inhibitors.
The IC50 for inhibition of decatenation can be visually assessed as the concentration of compound which leads to a 50% reduction in the mini-circle band. This is then verified using gel documentation software and statistical analysis. In the example below the inhibition of E. coli topo IV- catalysed decatenation of kDNA by ciprofloxacin is followed.