HTS kinesin ATPase Endpoint Assay Biochem Kit (BK053)

HTS kinesin ATPase Endpoint Assay Biochem Kit (BK053)

Product Uses Include

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

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. # KR01)
  3. Pre-formed microtubules (Cat. # MT002)
  4. Kinesin Reaction Buffer
  5. Paclitaxel (Cat. # 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 (see the CMW page). Of the assayed motor domains, only the ATPase activity of Eg5 was inhibited. (Fig 1)


 Figure 1. Inhibition of kinesin activity with the Eg5 specific inhibitor monastrol. The results show percent of full kinesin activity in the presence of 100 µM monastrol. As expected, only Eg5 ATPase activity is inhibited by monastrol. 


  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

For product Datasheets and MSDSs please click on the PDF links below.   For additional information, click on the FAQs tab above or contact our Technical Support department at

AuthorTitleJournalYearArticle Link
Wang, Wenjuan et al.The architecture of kinesin-3 KLP-6 reveals a multilevel-lockdown mechanism for autoinhibitionNature Communications 2022 13:12022ISSN 2041--1723
Aguilera, Cinthia et al.The Novel KIF1A Missense Variant (R169T) Strongly Reduces Microtubule Stimulated ATPase Activity and Is Associated With NESCAV SyndromeFrontiers in Neuroscience2021ISSN 1662-453X
Fukai, Ryota et al.Design, synthesis, and evaluation of a novel prodrug, a S-trityl-L-cysteine derivative targeting kinesin spindle proteinEuropean Journal of Medicinal Chemistry2021ISSN 1768-3254
Bae, Jinhye et al.A Micromachined Picocalorimeter Sensor for Liquid Samples with Application to Chemical Reactions and BiochemistryAdvanced Science2021ISSN 2198-3844
Tao, Li et al.Tum/RacGAP functions as a switch activating the Pav/kinesin-6 motorNature Communications2016ISSN 2041-1723
Reuter, Jason A. et al.Simul-seq: Combined DNA and RNA sequencing for whole-genome and transcriptome profilingNature Methods2016ISSN 1548-7105
Luo, Ruibai et al.Direct functional interaction of the kinesin-13 family membrane kinesin-like protein 2A (Kif2A) and Arf GAP with GTP-binding Protein-like, ankyrin repeats and PH domains1 (AGAP1)Journal of Biological Chemistry2016ISSN 1083-351X
Carbajales, Carlos et al.Structure-based design of new KSP-Eg5 inhibitors assisted by a targeted multicomponent reactionChemBioChem2014ISSN 1439-7633
Holland, Jason P. et al.Synthesis and Evaluation of Biphenyl Compounds as Kinesin Spindle Protein InhibitorsChemistry & Biodiversity2013ISSN 1612--1880
Holland, Jason P. et al.Fluorinated quinazolinones as potential radiotracers for imaging kinesin spindle protein expressionBioorganic & medicinal chemistry2013ISSN 1464--3391
Funk, C. Joel et al.Development of high-throughput screens for discovery of kinesin adenosine triphosphatase modulatorsAnalytical Biochemistry2004ISSN 0003-2697

Question 1:  What is the detection range of the Kinesin ATPase End-Point Biochem Kit (Cat. # BK053)?

Answer 1:  The reaction can detect phosphate over a range of 1 μM – 50 μM Pi (equivalent to 0.1 nmoles – 5.0 nmoles Pi in 100 μl reaction volume).


Question 2:  Is the Kinesin ATPase End-Point Biochem Kit (Cat. # BK053) compatible with other kinesins sold by Cytoskeleton or only the human kinesin heavy chain protein included with the kit?

Answer 2:  Yes, the Kinesin ATPase End-Point Biochem Kit (Cat. # BK053) can be used with any of the other kinesin motor proteins sold by Cytoskeleton, Inc.  Please see the protein’s datasheet on our website ( for representative data detailing each motor protein’s ATPase activity using either this kit (Cat. # BK053) or our Kinesin ELIPA Biochem Kit (Cat. # BK060).



If you have any questions concerning this product, please contact our Technical Service department at