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Product Uses Include
This is a Fluorescence based motility assay. To perform this assay effectively you will require a video microscope system with a shutter mechanism for time lapse image acquisition. Kinesin or dynein motors are coupled to the surface of a mcroscope perfusion chamber (kinesin heavy chain motor domain is included in the kit as a positive control) and fluorescent microtubules are added. The motor-induced motility of the microtubules can be monitored and measured in a fluorescence microscope.
The kit contains sufficient materials for 25 assays. The following components are included:
The motility of microtubules in a kinesin heavy chain (Cat. # KR01) coated chamber was followed with time lapse microscopy (Fig. 1)
Figure 1. Microtubule Motility Assay. The field was selected to show a sparse area of MT coverage in order to allow clear visualization of MT motility. Each frame represents a 5 min time-point, from 0 to 15 min. The position of each MT at time zero is marked by an arrow. The arrow position remains identical in each frame to serve as a reference point for MT movement. To obtain an average MT velocity, we recommend measure motility of approximately 30 - 50 individual MTs.
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: At what speed does the kinesin motor move in this assay?
Answer 1: The recombinant kinesin motor provided with this motility assay moves very slowly (average of 0.5 μm/min) and motility will not be seen without the use of a time lapse video microscopy system set on 2 to 5 min per frame. Native or full length recombinant kinesin moves with a velocity of 30 - 40 μm/min.
Question 2: I have high fluorescent background. How can I reduce it?
Answer 2: There will be some noticeable background fluorescence. The background fluorescence results from unpolymerized tubulin and can be removed by passing the microtubules over a glycerol cushion (see the manual for a detailed protocol).
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