The overall goal of this procedure is to load Nanospheres onto microtubules, which are gliding on a surface coated with kinase in motors. This is accomplished by first coating the surface of a glass flow cell with casein solution. The second step of the procedure is to absorb kinesin motor proteins onto the flow cell surface.
The third step of the procedure is to add the microtubules. The final step of the procedure is to add the strept haver in molecules, which will bind into the microtubules, and then add the nanospheres. The nanospheres bind into the flow cell surface where they may get picked up by the microtubules.
Ultimately, one can observe how nanospheres are picked up by microtubules and are transported across the surface using fluorescence microscopy. Hi, I'm Elaine Jen Smith from the laboratory of Dr.Henry Hess and the Department of Material Science and Engineering at the University of Florida. Today we will show you a procedure for the inverted motility assay.
We use this procedure in our laboratory to study loading of cargo onto microtubules and movement of molecular shuttles. So let's get started. Prior to the start of this procedure, freshly prepare all stock solutions as described in the written portion of this video.
Once stock solutions have been made, begin micro tubial preparation by pipetting 25 microliters of growth solution into a 0.65 milliliter micro centrifuge tube. Then add 6.25 microliters of the growth solution to 20 micrograms of lyophilized biotinylated tubulin. Vortex the solution and place it in a heat bath at 37 degrees Celsius for 30 minutes to polymerize following incubation, dilute the solution 100 fold in Taxol Buffer and gently vortex.
This solution serves to stabilize the microtubules. Label the solution as empty 100 and store it at room temperature. Next, make a tenfold dilution of MT 100 in anti fade buffer and label this solution as MT 1000.
Then prepare the Kinesin motors by diluting the kinesin solution 20 fold in case in a TP buffer. Label the solution as kinase in 20 and store it on ice. Finally, make the streptavidin and nanosphere solutions.
First, add Alexa Fluor 5 6 8 labeled streptavidin to a concentration of 100 nano molars in Antifa Buffer. Label the solution as STV 100, then dilute nanospheres 5, 000 fold in Antifa Buffer and label this mixture as NS 5, 000. Store both of these solutions over ice.
Following solution preparation for the cargo loading assay, construct a flow cell using a cover slide and a glass cover slip separated by double-sided tape. First stick two strips of double-sided tape at the edges of a cover slide, leaving a gap of about one centimeter between the strips. Then lay a cover slip over the tape.
Apply pressure to the cover slip using the flat edge of a tweezer to make a seal. The constructed flow cell is approximately two centimeters long, one centimeter wide and 100 microns high, and has a volume of approximately 20 microliters. To begin inverted assay assembly.
Place the tip of the pipette between the cover slip and the slide. Then inject 0.5 milligrams per milliliter casein solution into the flow cell. This coating on the flow cell will allow kinesin to retain its functionality upon absorption.
Wait five minutes for the casein solution to coat. Next, absorb Kinesin onto the flow cell by introducing the kinase in 20 solution solutions are introduced into the flow cells by placing the tip of the pipette between the cover slip and the slide. Then the solution is injected into the flow cell from one side, while it is simultaneously wicked out the other side.
With filter paper, allow five minutes for the kinase in to absorb. Next, introduce the microtubules into the flow cell by pipetting the MT 1000 solution into the flow cell. Allow five minutes for microtubules to settle and attach to the kinase in motors following micro tubial attachment to kinase in motors.
Coat the microtubules with fluorescent strep TTR in by introducing the STV 100 solution into the flow cell. Allow five minutes for strep TTR in molecules to bind to the microtubules. Wash out the excess strep TTR in by introducing three 30 microliter washes of Antifa solution into the flow cell.
Finally, introduce the biotinylated polystyrene fluorescein nanospheres by pipetting the NS 5, 000 solution into the flow cell. The surface absorbs stationary. Nanospheres will collide with the moving microtubules and load onto them.
In this experiment, an eclipse TE 2000 U fluorescence microscope equipped with a 100 x oil objective and X site, one 20 lamp and an exon E-M-C-C-D camera is used. First, apply one drop of immersion oil over the objective. Then mount the flow cell on the microscope stage using the trixy filter cube.
Bring the micro tubules into focus, then switch to the fitzy filter cube to view the nanospheres. Next, open the and or I exon program and set the acquisition mode to kinetic. Use an exposure time of 0.5 seconds, a kinetic series length of 20 and a kinetic cycle time of six seconds.
Switch the microscope from eyepiece to camera and begin acquiring images. For the first four images, use the trixi cube to image the microtubules. For the next 12 images, switch to the Fitz filter cube to acquire images of the nanospheres.
Then switch back to the trixi filter cube. For the last four images to image the microtubules, the microtubules are observed using the Trissy filter cube, the filter cube is switched to fite To visualize the nanospheres here, nanospheres being transported by microtubules are identified by the arrows. Not all nanospheres have been picked up by microtubules.
Now the location of the nanospheres is shown 36 seconds later revealing that the nanospheres have shifted. When the microtubules are imaged again, it becomes apparent that the microtubules have also traveled across the field of view. These images demonstrate that the nanospheres have been displaced due to micro tubial shuttling.
We have just shown you how to perform the inverted motility assay. When doing this procedure, it's important to remember to switch the filter cubes while acquiring images so that both the microtubules and the nanospheres can be seen. So that's it.
Thanks for watching and good luck with your experiments.
