QuantiChromTM ATPase/GTPase Assay Kit (DATG-200)

ATPases and GTPases catalyze the decomposition of ATP or GTP into ADP or GDP and free phosphate ion. These enzymes play key roles in transport, signal transduction, protein biosynthesis and cell differentiation. BioAssay Systems’ QuantiChromTM ATPase/GTPase Assay Kit offers a highly sensitive method for determining ATPase/GTPase activities in a microplate format. Its proprietary formulation features a single reagent for accurate determination of enzyme activity in 30 min at room temperature. The improved malachite green reagent forms a stable dark green color with liberated phosphate, which is measured on a plate reader (600 - 660 nm).

1. Preparation of phosphate standards. Prepare 800 μL Premix solution containing 50 μM phosphate by mixing 40 μL 1 mM phosphate standard with 760 μL distilled water. Number the tubes. Dilute standards as shown in the following Table. Pipette 40 μL standard in duplicate into wells of a clear-bottom 96-well plate.

2. Perform a series dilution of enzyme in assay buffer. Set up 40-μL reactions and a control with no enzyme in separate wells. Incubate the reaction for desired period of time (e.g. 30 min).

3. Add 200 μL Reagent and incubate 30 min at room temperature. Please note: use of a multi-chanel pipettor is recommended. The Reagent terminates the enzyme reaction and generates color with the free phosphate produced in the enzyme reaction.

4. Read OD620nm on a plate reader.

5. Enzyme activity. Calculate DOD values by subtracting OD values in reaction and control wells. Choose an enzyme concentration that gives a DOD of 0.5 to 1, this will ensure that substrate hydrolysis (<10%) is within the linear kinetics of reaction. Compute the concentration of free phosphate produced from the standard curve. 1 Unit of activity is the amount of enzyme that catalyzes the production of 1 μmole of free phosphate per minute under the assay conditions.

To evaluate an inhibitor or perform HTS, use the optimal enzyme concentration determined above. Incubate enzyme and inhibitor first for a certain period of time, before adding the substrate. At the end of reaction, add 200 μL Reagent for phosphate determination.

The procedure is similar as in the 96-well plate assay, except that 20 μL standards or 20 μL reaction mixture (10 μL Assay Buffer, 5 μL 4 mM ATP, 5 μL enzyme) are mixed with 80 μL Reagent.

Materials. Use ultrapure ATP and GTP. The provided 2x assay buffer contains 40 mM Tris, 80 mM NaCl, 8 mM MgAc2, 1 mM EDTA, pH 7.5. Other buffers (Hepes, Mes, Mops) can be used. Assay is compatible with 1 mM DTT, 2mM b-mercaptoethanol, 0.5 mg/mL BSA and 5% DMSO.