Technical Data
A3999-23
ATPase/GTPase Assay Kit, BioAssay™
1 Kit
Kits and Assays Storage: 4°CShipping: RT
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.
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).

Key Features:
High sensitivity: detection of as little of 2 pmoles of free phosphate.
Fast and convenient: single reagent, homogeneous “mix-and-measure” assay allows quantitation of enzyme activity within 30 minutes.
Robust and amenable to HTS: detection at 620nm greatly reduces potential interference by colored compounds. Z’ factors of >0.7 are observed in 96-well and 384-well plates. Can be readily automated on HTS liquid handling systems.

Applications:
Determination of ATPase and GTPase activity.
Drug Discovery: high-throughput screen for ATPase or GTPase inhibitors.

Kit Contents: 200 Assays In 96-Well Plate
Reagent: 50ml Assay Buffer: 10ml
Standard: 1mL 1mM phosphate
Storage conditions. The reagents and standard are stable for one year when stored at 4°C.
Precautions: reagent contains 0.27 M H2SO4. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.
Important: All reagents must be brought to room temperature before use. Before each assay, it is important to check that enzyme preparations and assay buffers do not contain free phosphate. This can be conveniently done by adding 200ul of the Reagent to 40ul sample solution. The blank OD values at 620 nm should be lower than 0.3. If the OD readings are higher than 0.3, check phosphate level. Lab detergents may contain high levels of phosphate. Make sure that lab wares are free from contaminating phosphate after thorough washes.

Activity Determination In 96-Well Plate:
1. Preparation of phosphate standards. Prepare 500ul Premix solution containing 50uM phosphate by mixing 25ul 1mM phosphate standard with 475ul distilled water. Number the tubes. Dilute standards as shown in the following Table. Pipette 40ul standard in duplicate into wells of a clear-bottom 96-well plate.
No Premix+H2O
Final Vol
(ul)
Phosphate
Conc (uM)
pmoles Phosphate
in 40ul
1 200ul+0ul 200 50 2,000
2 120ul+80ul 200 30 1,200
3 60ul+140ul 200 15 600
4 0ul+200ul 200 0 0
2. Perform a series dilution of enzyme in assay buffer. Set up 40-ul reactions and a control with no enzyme in separate wells. Incubate the reaction for desired period of time (e.g. 30 min). Reaction Well
20ul Assay Buffer
10ul enzyme
10ul 4mM ATP or GTP
Control Well
20ul Assay Buffer
10ul H2O
10ul 4mM ATP or GTP
3. Add 200ul 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 [Pi] (uM) from the standard curve.
Enzyme Activity=[Pi] (uM) x 40ul ÷ (10ul x t ) (U/L)
40ul and 10ul are the reaction volume and the enzyme volume in the assay. t is the reaction time (e.g. 30 min). 1 unit of activity is the amount of enzyme that catalyzes the production of 1uMole of free phosphate per minute under the assay conditions.

Inhibitor Assay In 96-Well Plate:
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 200ul Reagent for phosphate determination.
Reaction Well
20ul Assay Buffer
5ul enzyme
5ul inhibitor
10ul 4mM ATP or GTP
Control Well
20ul Assay Buffer
10ul 4mM ATP or GTP
10ul Buffer/DMSO

Assays In 384-Well Plate:
The procedure is similar as in the 96-well plate assay, except that 20ul standards or 20ul reaction mixture (10ul Assay Buffer, 5ul 4mM ATP, 5ul enzyme) are mixed with 80ul Reagent.

General Considerations:
Materials. Use ultrapure ATP and GTP. The provided 2x assay buffer contains 40mM Tris, 80mM NaCl, 8mM MgAc2, 1mM EDTA, pH 7.5. Other buffers (Hepes, Mes, Mops) can be used. Assay is compatible with 1mM DTT, 2mM b-mercaptoethanol, 0.5 mg/mL BSA and 5% DMSO.
 Important Note: This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.
Intended for research use only. Not for use in human, therapeutic, or diagnostic applications.