Microtubules (MTs) serve as a substrate for kinesin and dynein motor proteins as well as contributing to the shape and division of cells by the interaction with anchoring proteins. Kinesins operate by utilizing the energy of ATP to hydrolysis, an activity that is greatly enhanced in the presence of MTs. A MT activated kinesin ATPase assay is therefore used as a test for the biological activity of pre-formed microtubules (Cat. # MT002). This substrate is particularly useful for MT stimulated Kinesin ATPase assays that are used to develop novel anti-mitotic compounds, because the MT product is highly quality controlled we have achieved batch to batch variability of less than 5%. Click on the datasheet below for more information.
MTs are quality controlled by using the Enzyme Linked Inorganic Phosphate ATPase (ELIPA) assay. The assay is available in kit form from Cytoskeleton Inc. (Cat. # BK060) (1). The motor protein used in this assay is Kinesin Heavy Chain motor domain (KHC; Cat. # KR01).
Pizzi et al., 2010. Evidences of new biophysical properties of microtubules. In: Artifical Neural Networks, Ed. Seoyun J. Kwon. Chapter 20. Nova Science Publishers: Hauppauge, NY.
Zhang et al., 2005. Development of a high-throughput robotic fluorescence-based assay for HsEg5 inhibitor screening. Analytical Biochemistry. v 345, pp 326-335.
Question 1: What are the benefits and pitfalls of using pre-formed microtubules?
Answer 1: Some benefits of using pre-formed microtubules (Cat. # MT001 or MT002) are that the researcher can be assured of having highly purified and active microtubules that have already been functionally tested by our in-house quality control team. These pre-formed microtubules are also ideal for those researchers new to the tubulin field who have not polymerized tubulin previously and may not appreciate the importance of all the factors (temperature, GTP, enhancers, tubulin concentration) that influence tubulin polymerization. Some pitfalls are that the pre-formed microtubules fall within a target length and are difficult to modify. Additionally, because the microtubules have been lyophilized, there may be denatured regions of the microtubules that could limit their function. We test the ability of microtubules to activate kinesin ATPase activity and we know they work well in this assay, but the microtubules may not work as well for kinesin motility assays.
Question 2: What size are these microtubules?
Answer 2: On average, the length of the pre-formed microtubules (Cat. # MT001 or MT002) is 2 microns and the diameter is 25 nm. Microtubules consist of 13 protofilaments and each micron of microtubule length is composed of 1650 heterodimers.
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