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Bacterial RNA polymerase makes the mRNA, tRNA and rRNA of the cell. The bacterial holoenzyme is built from several subunits with a total mass of about 400 kDa, and it is a target for the discovery of new antibacterial compounds.
Kit Components
| Component | RPAP100K |
|---|---|
| Buffer RP | 400 µl |
| 100 x DNA | 33 µl |
| 100 x NTP mix | 33 µl |
| 1000 x Pyrophosphatase | 5 µl |
| Phosphate detection dye (also sold separately as MicroMolar Phosphate Assay Reagent, BHT16300173) | 4500 µl |
All reagents except the RNA polymerase are included; the kit is set up for 100 E. coli RNA polymerase reactions in 384-well format.
Assay Principle
The kit follows RNA synthesis on a double-stranded DNA template by measuring the pyrophosphate released in the reaction. Pyrophosphatase in the reaction converts the pyrophosphate to phosphate, which is then read with a phosphate detection dye by absorbance at 650 nm; inhibitors of the polymerase lower the signal.
Specifications
| Assay Target | Bacterial RNA polymerase activity |
|---|---|
| Target Type | RNA Polymerase |
| Assay Type | Enzyme Activity Assay; Inhibitor Screening Assay |
| Detection Method | Colorimetric |
| Detection Wavelength | Absorbance 650 nm |
| Plate Format | 384-well plate |
| Instrument Required | Absorbance microplate reader |
| Assay Time | About 63 min (60 min reaction + 3 min detection) |
| 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.
- RNA Polymerase Pyrophosphate Assay (PDF, see Documents): Covers catalog # RPAP100K.
Safety & Handling
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