| Assay Method Information | |
| | Supercoiling Assay |
| Description: | Supercoiling assay was performed using the commercially available kit (DNA gyrase supercoiling assay kit: SAS4001) from Inspiralis Pvt.limited, Norwich, UK. The assay was performed in 1.5 mL eppendorf tubes at room temperature. According to the assay protocol, 1 U of S. aureus DNA gyrase was incubated with 0.5 µg of relaxed pBR 322 DNA in 30 µL reaction volume at 37oC for 30 min with 40 mM HEPES. KOH (pH 7.6), 10 mM magnesium acetate, 10 mM DTT, 2 mM ATP, 500 mM potassium glutamate, 0.05 mg/mL albumin (BSA). While the standard compound novobiocin was the positive control, 4% DMSO was considered as negative control. Subsequently, each reaction was stopped by the addition of 30 µL of Stop dye [40% sucrose, 100 mM Tris-HCl (pH 7.5), 1 mM EDTA and 0.5 mg/mL bromophenol blue] (8), briefly centrifuged for 45 sec and was run in 1% agarose gel in 1X TAE buffer (40mM Tris acetate, 2mM EDTA). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |