Biomod/2012/Columbia/KinesinKings/Materials and Methods
Rhodamine-labeled microtubules were polymerized using Rhodamine labeled tubulin prepared from bovine brain and supplied by Cytoskeleton. 20 µg of tubulin were combined with 4mM MgCl, .0024% DMSO, and .16mg/ml GTP in a pH 6.9 BRB80 buffer solution. The aliquot was covered with aluminum foil in order to prevent photobleaching of the rhodamine during polymerization. The microtubules were polymerized in a 37⁰C water bath for 30 minutes, before being diluted 100-fold in a BRB80 solution containing 10 µm paclitaxel.
Flow cells were created using double-sided tape separating two ozone cleaned microscope coverslips. A 35x50mm coverslip made up the bottom surface with a 22x22 coverslip as the top surface. The corresponding thickness of the double sided tape created a flow channel with a volume of approximately 20 µL.
Volumes of 20 µL were pipetted into a marked edge ensuring that the flow direction did not alternate throughout the entire experiment. Poly-L-Lysine, acquired from Sigma-Aldrich at a molecular weight of 70,000 daltons, was diluted in a BRB80 buffer solution to a concentration of 1mg/ml. The diluted solution of poly-l-lysine was flowed into the cell in order to adequately coat the top and bottom glass surfaces. After a period of 90 minutes, BRB80 was flowed into the cell in order to remove any extraneous poly-l-lysine that was unable to bind to the glass surfaces. The microtubule solution was then flowed through and allowed to interact with the poly-l-lysine coated glass slides.
The imaging of the flow cell was done using fluorescent optical microscopy. The 570 nm wavelength of the rhodamine labeling of the microtubules allowed for an optical exicitation of the rhodamine under a green light. Using standard fluorescent microscopy, the images were captured with the Andor iXon DV885-JCS-VP EMCCD camera, a Nikon microscope with 10X and 40X objectives, and Sutter Lambda-LS Xenon Arc Lamp System. We used the Olympus Fluoview FV1000 Confocal Microscope with the FV10-ASW software. The images were processed and analyzed using ImageJ.