ATPases & GTPases

HTS 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.

Cytoskeleton's products have been cited hundreds of times over the past 18 years. A select few are described here, for more citations on individual products please use the "Citations" tab on each individual product page.

 

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 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 reaction volume I can use for the end-point ATPase assay?

Answer 2:  For the CytoPhos Phosphate Assay Biochem Kit (Cat. # BK054), the smallest reaction volume is 100 µl with a half-area plate (180 µl well volume) as this will maintain the pathlength while allowing smaller reaction volumes to be used.  It is strongly recommended to use a half area well plate to increase the absorbance signal.  If a standard 96 well plate is used (300 μl well volume), we recommend using a 200 μl final volume.  

 

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