Protein construct: Wild-type S. aureus RNA Polymerase purified from S. aureus.
MW: 400 kDa
Enzyme concentration: 2 µM
RNA polymerase assay: The RNA polymerase activity is measured by using the S. aureus RNA Polymerase Assay kit (Catalog No. RPA100KS).
Storage temperature: -20 or -80°C. Do not freeze-and-thaw repeatedly.
Enzyme dilution: Use the 1 x assay to dilute the enzyme just before the assay. Do not store diluted enzyme solution.
Assay Protocol
The following assay protocol is based on the 384-well plate assay format (plate type: Matrix 4318 or alike). The reaction volume is 30µl and the final assay volume is 60µl. For 96-well plate assays (plate type: Costar 3915 or alike), the reaction volume is 60µl and the final assay volume is 120µl.
1. Reagent preparation:
(1) 10 x DNA template: dilute the 100 x DNA template10-fold with water.
(2) 10 x enzyme: dilute the 100 x RNA polymerase 10-fold with the 1 x Buffer.
(3) 10 x NTP mix: dilute the 100 x NTP mix (50 mM) 10-fold with water.
(4) 1 x fluorescence dye: dilute the 10 x fluorescence dye 10-fold with water.
Note: The final concentration of S. aureus RNA polymerase in the assay is 25 nM. The assay buffer is 42.5 mM HEPES, pH 7.5, 42.5 mM NH4Cl, 2 mM DTT, 4 mM MgCl2, 0.005% Tween-20.
2. Reaction:
The total volume of each reaction mixture is 30µl including 18µl of H2O, 3µl of 10 x Buffer, 3µl of 10 x DNA template, 3µl of 10 x enzyme, 3µl of 10 x NTP mix. Incubate the reaction mixture in a standard black 384-well plate (Matrix 4318) at 37°C for 60 min.
3. Detection:
Add 30µl of the 1 x fluorescence dye into the 30µl of the reaction mixture. Incubate for 5 min. Measure the fluorescence intensity at 535 nm using the excitation wavelength at 485 nm.
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