Question 1: How can I set up a drug screening assay with my kinesin protein?

Answer 1: Kinesin motor proteins use microtubules (MTs) as a substrate to orchestrate a wide range of kinetic events within a cell. They have been shown to move cargoes such as chromosomes and vessicles along MT tracks. Kinesins operate by utilizing the energy of ATP by hydrolysis, an activity that is greatly enhanced in the presence of MTs. To screen drugs that modulate the interactions between motor proteins and MTs, a MT-activated kinesin ATPase assay can be used as a test for the MT-activated ATPase activity of kinesins. Cytoskeleton developed two such assays, one end-point and one kinetic, that are useful for the discovery and optimization of kinesin modulators. Both assays measure inorganic phosphate (Pi) levels generated by microtubule-activated kinesin adenosine triphosphatase (ATPase) activity. These two kinesin ATPase biochem assay kits (Cat. # BK053 and BK060) provide MTs and kinesin heavy chain (KHC) protein along with the necessary buffers and reagents to measure Pi production as a means of screening drugs that modulate kinesin and/or MT functional interactions. These kits are useful for discovering kinesin inhibitors and activators (Cat. # BK053 and BK060) as well as determining V_max and K_cat values for a kinesin motor protein (Cat. # BK060).

Question 2: What kits are available to characterize a putative motor protein?

Answer 2: Motor proteins utilize microtubules as a substrate, meaning that motor proteins are one type of microtubule-associated protein (MAP). To confirm that the putative motor protein binds with microtubules, Cytoskeleton’s microtubule binding protein spin down assay kit (Cat. # BK029) is available. The protein to be evaluated will need to be visualized by either Coomassie Blue or silver staining or with an antibody to either the protein itself or a tag conjugated to the protein (his, myc, etc.). A non-hydrolyzable ATP analog, e.g. AMPPNP, is usually used at 5 or 10 mM to maintain microtubule attachment. Microtubules also activate the ATPase activity of motor proteins which can be measured with one of our kinesin ATPase assays (Cat. # BK053 or BK060). These kits are useful for discovering kinesin inhibitors and activators (Cat. # BK053 and BK060) as well as determining V_max and K_cat values for a kinesin motor protein (Cat. # BK060). Cytoskeleton, Inc. also offers a Biochem Kit (Cat. # BK027) which allows the visualization of motor protein motility using fluorescently-labeled microtubules.