P. aeruginosa Gyrase DNA Supercoiling Assay Kit Plus

SKU:BHT16300071
Overview
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Fluorescence-based assay kit for P. aeruginosa DNA gyrase supercoiling activity and inhibitor screening. The H19 dye signal rises as relaxed DNA becomes supercoiled, read at Ex 485 nm / Em 535 nm in 96-well or 384-well plates. P. aeruginosa gyrase included; one 100-assay size.
Assay Target P. aeruginosa gyrase supercoiling
Detection Method Fluorescence
Detection Wavelength Ex 485 nm / Em 535 nm
Plate Format 96-well or 384-well plate
Assay Time About 65 min
Options selector
Catalog no. Size
DSA100KP 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 P. aeruginosa
Applications
  • Enzyme Activity
  • Compound Profiling
Assay type
  • Enzyme Activity Assay
  • Inhibitor Screening Assay
Assay target P. aeruginosa gyrase 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 or 384-well plate
Assay time
  • About 65 min
Storage Please contact us to confirm storage conditions.
Shipping Shipped frozen.
Catalog no. (Mfr.) DSA100KP
Main SKU BHT16300071
Drug Target Assay Kit

Bacterial DNA topoisomerase II (gyrase) converts relaxed circular DNA into supercoiled DNA (the DNA supercoiling reaction). The change in fluorescence is used to measure gyrase supercoiling and for high-throughput screening of gyrase inhibitors.

Kit Components

Component DSA100KP
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 P. aeruginosa gyrase 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 20 nM gyrase. Magnesium is essential; EDTA should be avoided. A reaction without ATP can serve as negative control.

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 P. aeruginosa gyrase supercoiling
Target Type DNA Topoisomerase II (Gyrase)
Organism P. aeruginosa
Assay Type Enzyme Activity Assay; Inhibitor Screening Assay
Detection Method Fluorescence
Detection Wavelength Ex 485 nm / Em 535 nm
Plate Format 96-well or 384-well plate
Instrument Required Fluorescence microplate reader
Assay Time About 65 min (60 min reaction + 5 min dye incubation)
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.

  • P. aeruginosa Gyrase DNA Supercoiling Assay (PDF, see Documents): Covers catalog # DSA100KP.

Safety & Handling

For Research Use Only (RUO). Not for use in diagnostic or therapeutic procedures. The P. aeruginosa Gyrase DNA Supercoiling 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 P. aeruginosa gyrase by the rise in H19 dye fluorescence as relaxed plasmid DNA becomes supercoiled. It is used to screen gyrase inhibitors.
Q.What is included in the kit?
A.The P. aeruginosa Gyrase DNA Supercoiling 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 P. aeruginosa gyrase (50 µl).
Q.Is a protocol available?
A.Yes. The manufacturer's protocol (P. aeruginosa Gyrase DNA Supercoiling Assay, linked under Documents) covers catalog # DSA100KP.
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 P. aeruginosa Gyrase DNA Supercoiling Assay Kit Plus is shipped frozen. Please contact us for long-term storage conditions.

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