Quinolone-resistant E. coli Gyrase Mutant D87G Assay Kit Plus

SKU:BHT16300072
Overview
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Fluorescence-based assay kit for the DNA supercoiling activity of quinolone-resistant E. coli gyrase mutant D87G, used to test new quinolone compounds. The H19 dye signal is read at Ex 485 nm / Em 535 nm in 96-well plates. Mutant gyrase included; one 100-assay size.
Assay Target E. coli gyrase D87G supercoiling
Detection Method Fluorescence
Detection Wavelength Ex 485 nm / Em 535 nm
Plate Format 96-well plate
Assay Time About 65 min
Options selector
Catalog no. Size
D87G100KE 100 assays
Available Options

Select the variant that best fits your experiment. Availability and lead time may vary by option.

  • Options: Size: 100 assays
  • Lead time: options listed in "Availability Content"; other statuses may take longer.
  • Storage: Please contact us to confirm storage conditions.
  • Shipping: Shipped frozen.
  • Upon receipt: store at recommended temperature as soon as possible.
  • Sales terms and conditions: Please review prior to ordering.
Field Specification
Organism E. coli
Applications
  • Enzyme Activity
  • Compound Profiling
Assay type
  • Enzyme Activity Assay
  • Inhibitor Screening Assay
Assay target E. coli gyrase D87G supercoiling
Assay area Antibacterial Drug Discovery
Target type DNA Topoisomerase II (Gyrase)
Detection method
  • Fluorescence
Detection wavelength Ex 485 nm / Em 535 nm
Instrument required Fluorescence microplate reader
Plate format 96-well plate
Assay time
  • About 65 min
Storage Please contact us to confirm storage conditions.
Shipping Shipped frozen.
Catalog no. (Mfr.) D87G100KE
Main SKU BHT16300072
Drug Target Assay Kit

Quinolones such as ciprofloxacin are widely used antibiotics that target bacterial DNA topoisomerases. The S83W and D87G mutations in the gyrA subunit of E. coli gyrase cause quinolone resistance, so new molecules that inhibit the mutant enzymes are needed.

This kit measures the DNA supercoiling activity of the quinolone-resistant D87G gyrase mutant, so it can be used to test the inhibitory activity of new quinolone compounds against it.

Kit Components

Component D87G100KE
10 x Buffer T2 600 µl
10 x Relaxed DNA 405 µl
1500 x H19 dye 20 µl
10 x ATP 450 µl
10 x H19 dilution buffer 3000 µl
100 x E. coli gyrase mutant D87G 50 µl

Final reaction conditions: 20 mM Tris-HCl pH 8, 35 mM NH4OAc, 4.6% glycerol, 1 mM DTT, 0.005% Brij35, 8 mM MgCl2, 25 µg/ml relaxed plasmid DNA, 1 mM ATP and 50 nM gyrase. Magnesium is essential; EDTA should be avoided.

Assay Principle

Relaxed and supercoiled DNA give different fluorescence with the H19 dye: in the presence of magnesium, relaxed DNA suppresses the dye signal much more than supercoiled DNA. As gyrase converts relaxed plasmid into its supercoiled form, fluorescence (Ex 485 nm / Em 535 nm) rises; inhibitors keep the signal low.

Specifications

Assay Target E. coli gyrase D87G supercoiling
Target Type DNA Topoisomerase II (Gyrase)
Organism E. coli
Assay Type Enzyme Activity Assay; Inhibitor Screening Assay
Detection Method Fluorescence
Detection Wavelength Ex 485 nm / Em 535 nm
Plate Format 96-well plate
Instrument Required Fluorescence microplate reader
Assay Time About 65 min (60 min reaction + 5 min dye incubation)
Mutation D87G in the gyrA subunit (quinolone-resistant)
Size 100 assays
Storage Please contact us to confirm storage conditions.
Shipping Shipped frozen.

Protocol

The manufacturer's protocol describes the procedure and reaction volumes.

  • Quinolone-resistant E. coli Gyrase Mutant Assays (PDF, see Documents): Covers catalog # D87G100KE.

Safety & Handling

For Research Use Only (RUO). Not for use in diagnostic or therapeutic procedures. The Quinolone-resistant E. coli Gyrase Mutant D87G Assay Kit Plus is shipped frozen. Follow standard laboratory safety practices when handling the reagents. Please contact us to confirm storage conditions.
Q.What does this kit measure?
A.It measures the DNA supercoiling activity of the quinolone-resistant E. coli gyrase mutant D87G (gyrA subunit) by H19 dye fluorescence, so compounds can be tested for inhibition of the resistant enzyme.
Q.What is included in the kit?
A.The Quinolone-resistant E. coli Gyrase Mutant D87G Assay Kit Plus (100 assays) includes 10 x Buffer T2 (600 µl), 10 x relaxed DNA (405 µl), 1500 x H19 dye (20 µl), 10 x ATP (450 µl), 10 x H19 dilution buffer (3000 µl) and 100 x E. coli gyrase mutant D87G (50 µl).
Q.Is a protocol available?
A.Yes. The manufacturer's protocol (Quinolone-resistant E. coli Gyrase Mutant Assays, linked under Documents) covers catalog # D87G100KE.
Q.How long does the assay take?
A.About 65 min (60 min reaction + 5 min dye incubation). See the protocol for the exact steps.
Q.How should the kit be stored?
A.The Quinolone-resistant E. coli Gyrase Mutant D87G Assay Kit Plus is shipped frozen. Please contact us for long-term storage conditions.

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