Microtubule (MT)/Organelle Motility Assays
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Dilute the axonemes with PM Buffer to the proper dilution (determined after preparation, see support protocol 2 below) and prepare 6x membranes (determined after preparation, see support protocol 4 below) by diluting organelles with PM buffer containing 1 mM GTP.
5ul 6x Golgi or ER membranes
1.5 ul of 20 x energy regeneration system
10 ul of 45 uM tubulin
1 ul of 15 mM MgGTP
12.5 ul of cytosol
Be careful to avoid introducing large bubbles into the chamber.
Slowly pipette 10 ul of PM buffer against one end of the perfusion chamber, while simultaneously wicking excess buffer from the opposite side of the chamber with the tip of a square of filter paper. Repeat this 2 more times.
It is difficult to focus on axonemes on the surface of the coverslip because of their very small size and the very bright illumination needed for VE-DIC. Align the slide on the stage so that an edge of the double stick tape forming the perfusion chamber perfectly bisects the area illuminated by the microscope condenser lens. Immerse the 100 x objective lens in oil, and focus on the edge of the tape. When you have brought the edge of the tape into view, back off fine focus until the very edge of the tape just begins to go out of focus. If you move the slide so the lens is within the area coated with axonemes, you should now be quite close to focus and the axonemes should be found with little difficulty.
Note the difference between the plus (longer, faster growing MTs) and minus (shorter, slower growing MTs) ends of the axonemes.
Note that often it takes up to 45 min for motility to develop. This time period is proportional to room temperature