This protocol is significant because it opens the door to further investigation in the design of nanoscale biological systems that are in dynamic equilibrium. This technique fills part of the gap between engineered and natural structures because it enables the study of what we could call, self-healing'or the dynamic replacement of molecular components. The most important advice for trying this technique for the first time is making sure that the experimenter's following all safety protocols.
First, prepare the stock solutions as outlined in the text protocol. Pipette 21.8 microliters of BRB80 Buffer into a small micro centrifuge tube. Add 1 microliter of the magnesium chloride stock solution, 1 microliter of the GTP stock solution, and 1.2 microliters of the DMSO stock solution to finish preparing the microtubule growth buffer.
To begin microtubule polymerization, add 6.25 microliters of microtubule growth buffer directly into a 20 microgram aliquot of lyophilized tubulin;labeled to be excited at 647 nanometers. Vortex at 40 RPS for 5 seconds. Cool the aliquot on ice for 5 minutes.
Then, incubate at 37 degree Celsius for 45 minutes. To begin microtubule stabilization, add 5 microliters of the aliquoted paclitaxel solution to 490 microliters of BRB80 buffer. Vortex the solution at 30 RPS for 10 seconds.
Once the 45 minutes of incubation for the microtubules is up, add 5 microliters of that solution to the BRB80, and paclitaxel mixture, to create the MT100 solution. First, add 9.0 microliters of the aliquoted casein solution to 291 microliters of BRB80 buffer. Pipette 83 microliters of the BRB80 casein solution into a new 6 milliliter micro centrifuge tube.
Add 1 microliter of D-glucose, glucose oxidase, catalase, DTT, creatine phosphate, and phosphokinase. Then, add 1 microliter of the stock ATP solution to the motility solution. Flick, or vortex the the aliquot to homogeneously distribute the chemicals.
Next, add 1 microliter of prepared kinesin solution to the motility solution, such that the final concentration is 20 nanomolar. Add 10 microliters of the MT100 solution to the motility solution. For flow cells, use both a large coverslip and a small coverslip.
Rinse all of the coverslips twice with ethanol and twice with ultrapure water. Sonicate the washed coverslips in ultrapure water for 5 minutes. Then, dry the coverslips in an oven at a temperature between 50 and 75 degrees Celsius.
Use a UV-ozone cleaner to treat one side of each coverslip for 15 minutes at room temperature and in normal atmospheric conditions. Using tweezers, flip each coverslip and use UV-ozone to treat the untreated side as well. Sonicate the treated coverslips in ultrapure water for 5 minutes.
Then, dry them in an oven at a temperature between 50 and 75 degrees Celsius. Next, don protective gear as outlined in the text protocol. Immerse each coverslip in saline solution for 15 seconds.
Wash the coverslips twice in toluene and three times in methanol. Use pressurized nitrogen to dry the coverslips. Once the coverslips are dry, cut a piece of double-sided tape that is 2 centimeters by 2.5 centimeters.
Cut this piece in half vertically into two 1 centimeter by 2.5 centimeter strips. Put the large coverslip on a delicate task wiper and stick the tape strips to it, length-wise along the edges to create a 1 centimeter by 2.5 centimeter area between the pieces of tape. Stick the small coverslip on top of the tape strips to finish the flow cell assembly.
First, flow approximately 20 microliters of the PEG-PPG-PEG solution into the assembled flow cell. Let the solution absorb on the surface for 5 minutes, then, exchange this solution with 20 microliters of BRB80 buffer three times by flowing the buffer in. Flow 20 microliters of the motility solution into the flow cell.
After this, seal the edges of the flow cell with grease to prevent evaporation if the planned experiment is longer than an hour. Perform the imaging using an objective-type total internal florescence microscopy. Place a drop of immersion oil on the objective.
Next, place the flow cell on the microscope platform. And bring the objective up until there is contact between the oil on the objective and the flow cell. Use the microscope's interlock cover system to block all laser light from escaping.
Then, turn on the laser and focus on the lower surface of the flow cell using a 642 nanometer laser to image the microtubules, and a 488 nanometer laser to image the GFP kinesin motors. Record the images or videos of interest. Images can be recorded for as long as there is motility in the flow cell.
In this study, an active nanoscale system, which self-assembles weakly binding building blocks to construct its own track, is presented. Using tirf microscopy, gliding microtubules are separately imaged from the kinesin motors. Microtubules are visible upon excitation with a 647 nanometer laser.
And the GFP kinesin are visible when excited with a 488 nanometer laser. The time between excitation and the red and green light was less than one second. As can be seen, gliding microtubules accumulate kinesin motors from solution and deposit them on the surface.
The kinesin motors remain in the wake of the microtubules for a short period of time before returning to solution. It is very important to have the correct concentration of reagents in the antifade of the motility solution, otherwise, photo bleaching would cause the experiment to fail. This technique paves the way for a more efficient use of protein motors in nanoscale engineer systems.
Thus, enabling the design and study of more complex nanostructures.
