Kits

Assays for kinesin motor proteins
Kinesins are a group of related molecular motors that utilize the energy of ATP hydrolysis to transport cargo such as chromosomes and vesicles along microtubule tracks. Functionally kinesins can be divided into two major groups, those involved in vesicle transport and membrane organization and those involved in mitosis. The central role of kinesins in cell division makes them of great interest as targets for anti-mitotics. Of critical importance to the premise of kinesins as drug targets is the fact that all available data indicates that antibodies and small molecules directed against mitotic kinesins specifically inhibit the mitotic process but have no effect upon kinesin vesicle transport functions. The fact that kinesins represent a new anti-mitotic target and that they are expressed predominantly during cell division suggests that they may be superior to and/or complementary to the current highly popular anti-tubulin drugs such as paclitaxel.

The Cytoskeleton Motor Werks™ range of products provides a comprehensive line of motor proteins , antibodies and assays that aid motor protein research.

Cytoskeleton offers a simple, colorimetric ATPase assay (Cat. # BK053). The assay measures microtubule (MT) activated kinesin motor ATPase activity. The simplicity of the assay lies in the fact that it uses a single step malachite based detection system and pre-formed MTs that require no preparation prior to the assay (Cat. # MT002). Another ATPase assay, Cat. # BK060, is a kinetic format and is very useful to measure the effects of inhibitors on microtubule stimulated kinesin ATPase. Both kits contain pre-formed microtubuleswhich are rigorously quality controlled and allow one to obtain high assay reproducibility. Kinesin motors are available separately in mg quantities and pre-formed MTs are also available separately in 2 mg, 10 mg and bulk sizes. In addition to mitotic motor proteins we provide several non-mitotic (vesicle transport) motors. Finally, the Microtubule Binding assay kit, Cat. # BK029, is useful to measure the binding affinity with microtubules and to determine factors that influence this equilibrium.

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 motor protein kits are shown below.  More citations are available on individual product pages.

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.

Kinesin ELIPA™ kit  (Cat. # BK060)

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.

Kinesin Motility Assay Biochem Kit (fluorescence format) (Cat. # BK027)

Abaza, A., Soleilhac, J. M., Westendorf, J., Piel, M., Crevel, I., Roux, A. and Pirollet, F. (2003). M phase phosphoprotein 1 is a human plus-end-directed kinesin-related protein required for cytokinesis. J. Biol. Chem. 278, 27844-27852.

Question 1:  How pure does my kinesin protein need to be to utilize the ATPase assays?

Answer 1:  There is no absolute purity value.  However, the purer the protein, the cleaner the assay will be.  We recommend titrating the kinesin of interest regardless of purity to achieve optimal results. We recommend beginning to titrate your protein between 0.2 μg and 1 μg per assay.  It is important to include control reactions in the assay, particularly if your kinesin of interest is in an impure state.  Important controls include: (i) reactions containing microtubules and ATP, minus kinesin protein and (ii) reactions containing your kinesin protein of interest and ATP, minus microtubules.

 

Question 2:  How do I measure the Kd affinity for microtubule binding to my protein of interest?

Answer 2:  The dissociation constant (Kd) is a means of assessing the binding affinity of two proteins for each other.  To determine the affinity of microtubules for a protein of interest, we recommend using a protocol modified from the methodology described in the microtubule binding protein spin-down assay biochem kit (Cat. # BK029).  Determination of the Kd measurement involves titrating either the test protein or the microtubules in the binding assay as described in the manual. The microtubule binding affinity for a given test protein can be defined in terms of the concentration of either the test protein or microtubules that is/are necessary to co-sediment 50% of the test protein.  For more detailed information, please see these references (Goode and Feinstein, 1994.  J. Cell Biol. 124, 769-782; Gustke et al., 1994. Biochemistry33, 9511-9522).

 

For more information, click on the Document tab above to see datasheets, or e-mail Technical Support at tservice@cytoskeleton.com.