| Assay Method Information | |
| | Enzyme Inhibition Assay |
| Description: | E. coli gyrase supercoiling and its inhibition was assayed using a kit procured from Inpiralis (K0001) and the protocol (PMID: 2172086) was adapted with necessary modifications. The compounds to be tested weree incubated for 10 minutes with 2.5 nM of E. coli DNA gyrase in a 30 μl reaction volume and 3.2% DMSO. The reactions were then started with the addition of 60 ng relaxed pBR322 plasmid DNA and continued for 45 min at 37° C. The reaction mixture contained 35 mM Tris.HCl (pH 7.5), 24 mM KCl, 1.8 mM spermidine, 4 mM MgCl2, 2 mM DTT, 6.5% (w/v) glycerol, 0.1 mg/mL BSA, and 1 mM ATP. The reaction was then stopped by addition of 0.75 L of Proteinase K (20 mg/mL) and 3 μL of 2% SDS and further incubated at 37° C. for 30 min. This was followed by the addition of 4 μL of STEB (40% (w/v) sucrose, 100 mM Tris-HCl pH8, 1 mM EDTA, 0.5 mg/ml Bromophenol Blue), and the supercoiled/relaxed forms of plasmid DNA were separated by agarose gel electrophoresis. The 1% agarose gels were run for 3 h at 4V/cm in 1×TAE (40 mM Tris, 20 mM Acetic acid, 1 mM EDTA). To visualize the DNA the gels were stained for 10 min with 0.7 g/mL ethidium bromide and excess dye was removed by several washes with water. IC50 were determined by quantifying the supercoiled and relaxed DNA in each of the reactions from a gel image by a densitometric method using the Quantity One Software (Bio-rad). |
| Affinity data for this assay | |
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