Description

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’ QuantiChrom^TM 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: 50 mL                                Assay Buffer: 10 mL 

Standard: 1mL 1 mM 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 200 μL of the Reagent to 40 μL 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 800 mL Premix solution containing 50 mM phosphate by mixing 40 mL 1 mM phosphate standard with 760 mL distilled water. Number the tubes. Dilute standards as shown in the following Table. Pipette 40 mL standard in duplicate into wells of a clear-bottom 96-well plate.