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Kinesin ELIPA Biochem Kit

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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 cargoes, 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 ELIPA™ (Enzyme Linked Inorganic Phosphate Assay) Biochem Kit.  The assay is an adaptation of a method originally described by Webb for the measurement of glycerol kinase plus D-glyceraldehyde ATPase activity and for actin activated myosin ATPase (4).  The assay is based upon an absorbance shift (330 - 360 nm) that occurs when 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) is catalytically converted to 2-amino-6-mercapto-7-methyl purine in the presence of inorganic phosphate (Pi).  The reaction is catalyzed by purine nucleoside phosphorylase (PNP).  One molecule of inorganic phosphate will yield one molecule of 2-amino-6-mercapto-7-methyl purine in an essentially irreversible reaction (5).  Thus, the absorbance at 360 nm is directly proportional to the amount of Pi generated in the kinesin ATPase reaction.

End point assays for kinesin MT activated ATPase activity in high throughput format can be performed with Biochem Kit™ BK053.  Cytoskeleton, Inc. also offers a wide range of kinesin motors (see the Cytoskeleton Motor Werks page).

 

Kit contents

The kit contains sufficient material for 96 assays. The following components are included:

  1. Reaction buffer
  2. ELIPA reagent 1 (MESG)
  3. ELIPA reagent 2 (PNP)
  4. Kinesin Heavy Chain motor domain protein (Cat nr KR01)
  5. Pre-formed microtubules (Cat nr MT001)
  6. Paclitaxel (Cat nr TXD01)
  7. Phosphate standard
  8. ELIPA reagent 1 resuspension buffer
  9. ATP stock (Cat nr BSA04)
  10. GTP stock (Cat nr BST06)
  11. Manual with detailed protocols and extensive troubleshooting guide

Equipment needed

  1. 96-well plate spectrophotometer capable at reading 360-370 nm. Monochromatic (360 nm) or filter based with narrow bandwith (370 nm filter with no more than 10 nm bandwith)

Example results

The microtubule activated ATPase activity of Kinesin Heavy Chain (Cat nrKR01), CENP-E (Cat nr CP01), Eg5 (Cat nr EG01) and Chromokinesin (Cat nr CR01) was measured with BK060.

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

 

Product Uses Include

  • Kinetic measurement of microtubule activated kinesin ATPase activity
  • Discovery of kinesin inhibitors
  • Measurement of kinesin inhibitor IC50s

References

  1. Vale, R. D. and Fletterick, R. J. (1997).  The design plan of kinesin motors. Ann. Rev. Cell Dev. Biol. 13: 745-777.
  2. Endow, S. A. (1999).  Microtubule motors in spindle and chromosome motility.  Eur. J. Biochem 262, 12-18.
  3. Johnson, K. A. and Gilbert, S. P. (1995).  Pathway of the microtubule-kinesin ATPase.  Biophys. J. 68, 173-179.
  4. Webb, M. R. (1992). A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems.  Proc. Natl. Acad. Sci. USA 89, 4884-4887.
  5. Cheng Q., Wang Z-X., and Killilea S. D. (1997). A continuous spectrophotometric assay for protein phosphatases. Anal. Biochem. 226, 68-73.

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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