ATPase/GTPase Assay Kit (Colorimetric) (BA0017)

Product Name:	ATPase/GTPase Assay Kit (Colorimetric) (BA0017)
SKU:	BA0017
Detection Method:	Colorimetric (OD 620 nm; malachite green)
Detection Range:	Detection of 0.007 U/L ATPase or GTPase activity; phosphate standards 0-50 µM
Sample Type:	['Enzyme preparations']
Species Reactivity:	All
Assay Time:	~30 minutes plus 30 min colour development
Kit Size:	200 Assays
Equipment Required:	Microplate reader
Storage:	4°C
Shelf Life:	12 months (stable for one year at 4°C)
Shipping:	Room Temperature

ATPases and GTPases catalyse 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. This kit offers a highly sensitive method for determining ATPase and 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 colour with liberated phosphate, which is measured on a plate reader (600-660 nm).

High sensitivity: detection of 0.007 U/L ATPase or GTPase activity.
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 620 nm greatly reduces potential interference by coloured compounds, Z' factors of >0.7 are observed in 96-well and 384-well plates, and it can be readily automated on HTS liquid handling systems.

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

Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Step	Procedure
1	Bring all reagents to room temperature. Before each assay, check that enzyme preparations and assay buffers do not contain free phosphate by adding 200 µL of Reagent to 40 µL sample solution; blank OD values at 620 nm should be lower than 0.3, and higher readings indicate phosphate contamination (for example from lab detergents).
2	Prepare phosphate standards: make 500 µL of a 50 µM phosphate Premix by mixing 25 µL of 1 mM phosphate standard with 475 µL distilled water, dilute the standards as shown in the standard curve table and pipette 40 µL of each standard in duplicate into wells of a clear-bottom 96-well plate.
3	Perform a serial dilution of the enzyme in assay buffer and set up 40 µL reaction wells (20 µL Assay Buffer, 10 µL Enzyme, 10 µL 4 mM ATP or GTP) alongside control wells with no enzyme (30 µL Assay Buffer, 10 µL 4 mM ATP or GTP). Incubate the reaction for the desired period (e.g. 30 min).
4	Add 200 µL Reagent to each well and incubate 30 min at room temperature; the Reagent terminates the enzyme reaction and generates colour with the free phosphate produced. Use of a multi-channel pipettor is recommended.
5	Read OD620nm on a plate reader.
6	Calculate the change in OD by subtracting control-well from reaction-well OD values, choosing an enzyme concentration that gives a change in OD of 0.5 to 1, and determine the free phosphate concentration from the standard curve.

Enzyme Activity = [Pi] (µM) x 40 µL / (10 µL x t) (U/L), where [Pi] is the concentration of free phosphate produced (from the standard curve), 40 µL and 10 µL are the reaction and enzyme volumes, and t is the reaction time (e.g. 30 min). 1 unit of activity is the amount of enzyme that catalyses the production of 1 µmole of free phosphate per minute under the assay conditions.

Component	Quantity	Storage
Reagent	50 mL	4°C
Assay Buffer	10 mL	4°C
Standard (1 mM phosphate)	1 mL	4°C

ATPase/GTPase Assay Kit (Colorimetric) (BA0017)

Description