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Kinesin HTS ATPase end point assay

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Introduction

Eukaryotic kinesin motor proteins orchestrate a wide range of kinetic events within a cell.  They have been shown to move cargo, such as chromosomes and vesicles, along microtubule tracks (1).  They also play a major role in the organization of cytoskeletal architecture as evidenced in the establishment of the microtubule spindle during mitosis (2). 

Kinesins operate by utilizing the energy of ATP hydrolysis to move along their microtubule (MT) substrates.  Once a kinesin motor binds to its MT track, the ATPase rate of the motor is often enhanced several hundred to several thousand-fold (3).  MT activated kinesin ATPase is a major parameter in  motor function and serves as a powerful method to monitor and study kinesin activity under various experimental conditions.

As part of its Cytoskeleton Motor Werks (CMW) line of research reagents, Cytoskeleton, Inc. has developed the Kinesin ATPase End-Point Biochem Kit™ (BK053). The kinesin end-point assay is an extremely quick and economical way to measure inorganic phosphate (Pi) generated during the microtubule activated ATPase activity of kinesin motor proteins.  Large numbers of assays can be performed simultaneously in a homogenous reaction, making the assay highly suitable for HTS applications. The assay is based upon a colorimetric change, measured at 650 nm.

More detailed kinetic studies for kinesin MT activated ATPase assays can be performed with Biochem Kit BK060.  Cytoskeleton, Inc. also offers a wide range of kinesin motors (see the Cytoskeleton Motor Werks page).

 

Kit contents

The kit contains sufficient materials for 1000 assays. The following components are included:

  1. CytoPhos™ reagent
  2. Kinesin motor protein (Cat nr KR01)
  3. Pre-formed microtubules (Cat nr MT001)
  4. Kinesin Reaction Buffer
  5. Paclitaxel (Cat nr TXD01)
  6. Phosphate standard
  7. DMSO
  8. Manual with detailed protocols and extensive troubleshooting guide

Equipment needed

  1. Spectrophotometer capable of measuring absorbance at 650 nm wavelength.
  2. Small capacity (100-1000 µl) cuvettes or 96-well microtiter plates.

Example results

The activity of the Eg5-specific inhibitor, monastrol, was tested on 9 different kinesin motor domains (find them all here). Of the assayed motor domains, only the ATPase activity of Eg5 was inhibited.

See the figure 1 in the Gallery, Related Download section.

 

Product Uses Include

  • Measuring microtubule-actived kinesin ATPase activity
  • Discovering/characterizing kinesin inhibitors
  • Discovering/characterizing kinesin regulating cofactors

 

References

  1. Goldstein, L. (1993) Annu. Rev. Genet. 27, 319-351
  2. Sawin, K. E. and Scholey, J. M. (1991) Trends Cell Biol. 1, 122-129
  3. Kuznetsov, S. A., and Gelfand, V. I. (1986)  Proc. Natl. Acad. Sci. USA 83, 8530-8534

Examples of publications where this product was used

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.

 


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