Gliding motility assay using 22063mM D2O in solution. Replicate 1/3. ROI 1/15.

2012-11-15T13:35:44Z (GMT) by Andy Maloney
<p>• This is a gliding motility assay using kinesin-1 and rhodamine labeled microtubules.</p> <p>• The buffer used was PEM (also known as BRB80) with the inclusion of 22063mM D2O.</p> <p>• Images were taken with an Andor Luca EMCCD camera on an Olympus IX-71 microscope using a 60x 1.42NA PlanApo objective.</p> <p>• This fileset is replicate 1/3 and ROI 1/15.</p> <p>• Images were taken to investigate speed variations of microtubules due to the inclusion of D2O in the motility assay. </p><p>• The temperature of the slide was held constant at 32°C by an objective heater. Early ROIs, i.e. 1-5, have slower giding speeds due to the increasing temperature of the slide which, was prepared at room temperature before observation. After ROI 5 the gliding speed stabilizes.</p> <p>• Pixels lengths were measured to be 166nm/pixel using a stage micrometer.</p> <p>• After the files have downloaded, a simple command line entry will convert the images to a movie. Mencoder must be installed. ls > list.txt | mencoder mf://@list.txt -mf w=496:h=658:fps=30:type=png -ovc lavc -lavcopts vcodec=mpeg4:mbd=2:trell -o output.avi</p> <p>• Upload of these files were made possible through the figshare API.</p>
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