ATPase & GTPase Assays

Assays for ATPases, GTPases or phosphate measurements
Many processes inside the cell are dependent on nucleotide hydrolysis, which results in inorganic phosphate (Pi) liberation. Measurement of Pi generation by ATPases & GTPases is a simple, cost effective functional assay for this class of important enzymes. Cytoskeleton, Inc. provides several NTPase assays for HTS applications. There is a choice of non-radioactive endpoint assay with a 1 µM phosphate limit of detection (BK054), which is good for moderate or high activity NTPases (Kcat >0.05). There is a radioactive filter based endpoint assay (BK055) with 10 nM detection limit that is good for small G-proteins and microtubule polymerizations, which have low GTPase activity (Kcat <0.01).

 

For more information about Phosphate Assays please see About Phosphate Assays.

Many publications cite the use of Cytoskeleton's kits in the Materials and Methods section of papers. Usually the citation is associated with a particular result in the form of a graph or image that helps the you, the authors, present your findings. This indicates the utility of the Kits to produce publication quality data in a short timeframe thus helping improve the productivity of your efforts. Example citations for phosphate assay kits are shown below.  More citations are available on individual product pages.

 

ATPase Kinetic ELIPA™ Assay Kit (Cat. # BK051)

Funk, C. J., Davis, A. S., Hopkins, J. A. and Middleton, K. M. (2004). Development of high-throughput screens for discovery of kinesin adenosine triphosphatase modulators. Anal. Biochem. 329, 68-76.

GTPase Kinetic ELIPA™ Assay Kit (Cat. # BK052)

Funk, C. J., Davis, A. S., Hopkins, J. A. and Middleton, K. M. (2004). Development of high-throughput screens for discovery of kinesin adenosine triphosphatase modulators. Anal. Biochem. 329, 68-76

HTS kinesin ATPase Endpoint Assay Biochem Kit  (Cat. # BK053)

Funk, C. J., Davis, A. S., Hopkins, J. A. and Middleton, K. M. (2004). Development of high-throughput screens for discovery of kinesin adenosine triphosphatase modulators. Anal. Biochem. 329, 68-76.

CytoPhos™ Endpoint Phosphate Assay (Cat. # BK054)

Funk, C. J., Davis, A. S., Hopkins, J. A. and Middleton, K. M. (2004). Development of high-throughput screens for discovery of kinesin adenosine triphosphatase modulators. Anal. Biochem. 329, 68-76.

Question 1:  My GTPase has a low GTPase activity.  Can I still use it in an assay?

Answer 1: Yes, Cytoskeleton offers the EasyRad Phosphate Assay Biochem Kit (Cat. # BK055) that is specifically designed for low activity GTPases.  The kit is a simple two step assay based on the separation of 32 or 33Phosphate from 32 or 33Gamma-Phosphate-Nucleoside Triphosphate (NTP).  This is the highest sensitivity phosphate assay available, allowing Kcat measurements as low as 0.00001 (NTPs hydrolysed per sec per molecule). 

 

Question 2:  What is the smallest amount of test protein that I can use in the kinetic ATPase assay (Cat.# BK051)?

Answer 2: We recommend preparing the test protein at a concentration of 1 mg/ml if purity is >90%.  If the protein is at lower purity, we recommend increasing the total protein concentration to compensate for the lower purity. The protein must be in a phosphate-free buffer such as Tris-HCl (PBS is not suitable).  We strongly recommend performing a protein titration. A good titration range is between 0.1 μg – 20 μg per assay.

 

For more information, click on the Documents tab above and see the datasheets, or contact Technical Support at tservice@cytoskeleton.com.