HTS assays for 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.
For HTS applications, 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). The rigorously quality controlled MTs also account for 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 available. These are suitable as controls in secondary screens such as Cat. # BK060. The panel of kinesins available are representative of the major classes of kinesin proteins and therefore offer an excellent screening panel for target specificity.
Motor proteins are in the large family of ATP binding proteins, which also includes kinases and DNA polymerases among others. Well characterized ATP analog libraries can therefore be used as a starting point for discovering inhibitors of the kinesin ATPase proteins. Such a familial approach to drug discovery promises to be efficient and highly cost effective yielding high quality hits. More information can be found in a review of the use of ATP analog libraries (Chene, P. Expert Opin. Ther. Targets (2003) 7: 453-461).
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.
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|
Question 1: Which assay format (kit) is best suited for screening 5000 compounds?
Answer 1: Cytoskeleton offers two Kinesin ATPase Assay Biochem Kits (Cat. # BK053 and BK060). For an initial screening, we recommend the Kinesin ATPase End-point Biochem Kit (Cat. # BK053) which offers 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. More detailed kinetic studies for microtubule-activated kinesin ATPase activity can be performed with the Kinesin ELIPA Biochem Kit (Cat. # BK060). Both kits come with the necessary reagents and buffers to measure the ATPase activity of kinesins, including pre-formed microtubules, a kinesin heavy chain motor domain protein as a positive control and a phosphate standard. Cytoskeleton offers a wide range of kinesin motor proteins that can be used with these assays.
Question 2: How pure does my kinesin protein need to be to utilize the ATPase assays?
Answer 2: 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 3: Can I get a quote for Cytoskeleton to run some kinesin ATPase assays?
Answer 3: Yes, please contact Technical Support at email@example.com or 303-322-2254 to talk to a scientist about our custom screening services including kinesin ATPase assays.
For more information, click on the Document tab above to see datasheets, or e-mail Technical Support at firstname.lastname@example.org.
Heavy Meromyosin Protein (HMM Fragment from cardiac muscle tissue)