By Jennifer Ross, Wallace F. Marshall
This new quantity of Methods in mobilephone Biology seems to be at construction a mobile from its part elements. Chapters conceal such issues as engineering motor scaffolds, synthetic cytoskeletons, interconnected droplet networks and synthetic cells; construction cytoskeletal structures and synthetic actin cortex on pillar arrays; reconstituting membrane fission, actin bundles at membranes, actin cortex in droplets, dynein-dynactin mediated shipment shipping, brain platforms, protein-lipid machines that remodel membranes, protein interactions and signaling on synthetic lipid bilayers. With state of the art fabric, this finished assortment is meant to steer researchers for years to come.
- Covers sections on version platforms and sensible experiences, imaging-based methods and rising studies
- Chapters are written by means of specialists within the field
- Cutting-edge material
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Extra info for Building a cell from its component parts
Assaying processive movement of kinesin by fluorescence microscopy. Methods in Enzymology, 298, 154e171. , Kubiak, J. , Nedelec, F. , & Gueroui, Z. (2009). Effects of confinement on the self-organization of microtubules and motors. Current Biology: CB, 19, 954e960. , & Ross, J. L. (2013). Microtubule organization by kinesin motors and microtubule crosslinking protein MAP65. Journal of Physics. Condensed Matter: an Institute of Physics Journal, 25, 374103. Ross, J. , & Dixit, R. (2010). Multiple color single molecule TIRF imaging and tracking of MAPs and motors.
Go to plugins and select the Multiple Kymograph plugin. html. In the pop-up window, it will ask what width to integrate over perpendicular to the line segment that was drawn. We typically use three pixels, since that will cover the diffraction-limited width of the image of the microtubule. This will create the kymograph in a new window (Figure 2(E)). Multiple kymograph allows one to create many kymographs simultaneously, as outlined in the documentation for the plugin. The new frame has x and y axes whose orientation is not intuitive.
5. Flow in 10 mL of microtubule dilution into the flow chamber. Incubate for 2 min. 6. During the 2 min incubation add glucose and deoxy to the Motility Mix buffer. 7. Flow in 10 mL of Motility Mix buffer to the flow chamber. The Motility Mix activates the gliding assay by giving kinesin the ATP source to move the microtubules. 8. Image using epifluorescence. Take movies with 3e5 s between frames, shuttering in between to avoid photobleaching or photodamage. Movies are saved as stacks of tiffs or ND2 files using Nikon Elements software.