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<div id="nav"> <h1 id="sitename"><a href="http://openwetware.org/wiki/Biomod/2012/Columbia/KinesinKings">Kinesin Kings</a></h1> <u1 id ="menu"> <ul class="blockeasing"> <li class="main"><a href="http://openwetware.org/wiki/Biomod/2012/Columbia/KinesinKings/AboutUs" onmouseover="this.style.borderBottomColor ='#aaaaaa'"

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                             <li class="main"><a href="http://openwetware.org/wiki/Biomod/2012/Columbia/KinesinKings/Overview">Overview</a>

<ul class="subnav"> <li><a href="http://openwetware.org/wiki/Biomod/2012/Columbia/KinesinKings/Overview#Objectives">Objectives</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/Columbia/KinesinKings/Overview#Abstract">Abstract</a></li> <!-- <li><a href="#">LinkedIn</a></li> --> <!-- <li><a href="#">Facebook</a></li> --> </ul> </li>

                             <li class="main"><a href="http://openwetware.org/wiki/Biomod/2012/Columbia/KinesinKings/Background">Background</a>

<ul class="subnav"> <li><a href="http://openwetware.org/wiki/Biomod/2012/Columbia/KinesinKings/Background#Definitions">Definitions</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/Columbia/KinesinKings/Background#Current_Practices">Common Practices</a></li> <!-- <li><a href="#">Scientific Knowledge</a></li> -->

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testing text


Materials

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.


Methods

Experiments

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.


Imaging

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.


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  2. menu li a {display:block; /*float:right; padding:15px 15px 0 15px;*/ text-decoration:none; color:#85c2e3; text-shadow:#000 1px 1px 1px;}
  3. menu li a:visited, #menu li a:active {text-decoration:none; color:#85c2e3; text-shadow:#000 1px 1px 1px;}
  4. menu li a:hover {color:#fff; font: 13px bold "Helvetica", Arial, Helvetica, sans-serif;}
  5. menu li.active a{color:#fff;}

/*#menu li a span {text-decoration: underline;!important;}**/ .post { padding:8px; margin-bottom:10px;} .columns {padding:8px;} .columnleft {float:left; width:49%;} .columnright {float:right; width:49%;} .col {float:right; width:41%; margin-right:12px; margin-bottom:15px;} .col p {margin-bottom:10px!important;} .txtbutton a{display:block; background:url(images/learnmore.jpg) no-repeat!important; height:19px; width:90px; text-align:center; font:bold 12px "Trebuchet MS", Arial, Helvetica, sans-serif; color:#ccc!important; text-shadow:#000 1px 1px 1px; text-decoration:none; padding:2px 0 0 0!important;} .txtbutton a:visited, .txtbutton a:active {display:block; background:url(images/learnmore.jpg) no-repeat!important; height:19px; width:90px; text-align:center; font:bold 12px "Trebuchet MS", Arial, Helvetica, sans-serif; color:#ccc!important; text-shadow:#000 1px 1px 1px; text-decoration:none; padding:2px 0 0 0!important;} .txtbutton a:hover {background:url(images/learnmore-hover.jpg) no-repeat!important; color:#fff!important;}

  1. bottombar {

background:#000;width:770px; position:fixed; bottom:0; left:50%; height:25px; margin:0 0 0 -385px; z-index:998; }

.categories li {display:block; height:28px; font: bold 16px "Trebuchet MS", Arial, Helvetica, sans-serif;} .categories li a {display:block; background:url(images/catgory1.jpg) no-repeat; width:254px; height:22px; padding:4px 10px 0 10px; text-shadow: #000 1px 1px 1px; color:#555;} .categories li a:visited, .categories li a:active {color:#555;} .categories li a:hover {background:url(images/catgory2.jpg) no-repeat; color:#fff;}

  1. sidebar ul {margin-bottom:10px;}

.block { width:274px; margin-bottom:10px;} .blocktop{background: #ffffff /*url(images/darkbox1.jpg) no-repeat; height:6px;*/} .blockmid {padding:8px; background: #ffffff/*url(images/darkbox2.jpg) repeat-y;*/} /*.blockfooter {background:url(images/darkbox3.jpg) no-repeat; height:6px;}*/ p.ads {display:block;} img, a img {border:none;} p.ads img {display:block; float:left; margin:2px;} .breadcrumb {display:block; width:630px; float:left; padding:12px 0 0 10px;} .breadcrumb li { display:inline; font:bold 12px "Trebuchet MS", Arial, Helvetica, sans-serif; text-shadow: #000 1px 1px 1px;}

  1. searchbar{margin-left:665px; padding:6px 0 0 0;}
  2. searchbar div { float:left; margin-right:5px;}
  3. searchbar input {color:#fff;}
  4. searchbar .keyword {display:block; background: transparent url(images/searchfield.jpg) no-repeat; width:185px; height:27px; padding:5px 5px 0 5px; border:none; float:left; color:#fff;}
  5. sidebar .posts {padding:5px;}
  6. sidebar .posts li {display:block; padding:5px 0 5px 0;}
  7. sidebar .posts li span {font:11px "Trebuchet MS", Arial, Helvetica, sans-serif; color:#444; display:block;}
  8. sidebar .posts li a {display:block; border-bottom: dashed 1px #9EC5DA;}
  9. footerlinks {background:url(http://openwetware.org/images/6/60/Bottombar.jpg) repeat-x; height:41px;}
  10. bottom {background:#000; padding:5px;}

.alignleft {float:left;} .alignright {float:right;} .post .articleimg {padding:8px; background:#111; border:solid 1px #000;} img.alignleft { margin:0 15px 0 0;} img.alignright {margin:0 0 0 15px;} .post a {background:#171612; text-shadow:#000 1px 1px 1px; } .post a:hover {background:#000; color:#fff;}

  1. footernav {display:block; padding:5px 25px 5px 5px;}
  2. footernav li { display:inline; font:bold 12px "Trebuchet MS", Arial, Helvetica, sans-serif; text-shadow: #000 1px 1px 1px; padding:0px 0px 0px 0px;}

.credit {font:11px "Trebuchet MS", Arial, Helvetica, sans-serif; color:#444; display:block; padding:5px 0 5px 0;} h1 a, h2 a, h3 a, h4 a {background:none!important;}

  1. comments {padding:10px; background: #171612;}
  2. comments h2 {background:#000; padding:10px; font-size:18px!important;}
  3. comments .entry {padding:10px; background:#11100D; margin-bottom:10px;}
  4. comments .entry p {display:block; margin-bottom:8px!important;}
  5. comments .entry .avt {float:left; width:55px; margin-right:10px;}

.avt img {background:#000; padding:5px;}

  1. comments .entry .con, #comments .entry .name, #comments .entry .date { margin-left:70px;}
  2. comments .entry .con blockquote {font:italic 14px Georgia, "Times New Roman", Times, serif; color:#666; padding-left:15px!important; border-left: solid 5px #333!important; margin:0!important;}

h3#respond {color: #FC6;} .comments-form {background:#000; padding:10px;} .formid, .formuri, .formemail {padding:3px; height:25px; background:#171612; border:solid 1px #1C1B17; width:250px; color:#999; font-size:16px; } .comments-form label {padding-left:15px;} .comments-form textarea {padding:3px; background:#171612; border:solid 1px #1C1B17; color:#999; font-size:16px; } .postheader {margin-bottom:18px;} .postheader .postdetails {font-size:11px; color:#555;} .post blockquote {display:block; margin-left:10px; padding:5px 5px 5px 40px; border-left: solid 5px #333; font:14px italic Georgia, "Times New Roman", Times, serif; background:url(images/quote.jpg) 5px 0 no-repeat; margin-bottom:18px;} cite {display:block; color:#aaa; font-weight:bold;} .post blockquote p {margin-bottom:0!important;} blockquote.alignleft {width:200px;} blockquote.alignright {width:200px;} .post ul, .post ol {margin-bottom:18px;} .post ul li {display:block; padding:3px; line-height:16px; background:url(images/bullet.gif) 5px 5px no-repeat; padding-left:20px; margin-bottom:10px;} .post ol { margin-left:25px;} .post ol li { padding:3px; line-height:16px; margin-bottom:10px;} .post code {display:block; white-space:pre; background:#171612; padding:5px; margin-bottom:18px; font:12px "Courier New", Courier, monospace; border: solid 1px #312F29;}

.tags {padding:8px; background:#171612; margin-bottom:18px;}

.authorinfo {padding:8px; border-top: dashed 1px #333; border-bottom: dashed 1px #333; margin-bottom:18px;} .authorinfo img {float:left; margin-right:15px;} .postinfo {font-size:11px; font-weight:bold; display: block; margin-bottom:18px; font-family:"Trebuchet MS", Arial, Helvetica, sans-serif;} .postbottom { border-bottom:dashed 1px #000; margin-bottom:18px; padding-bottom:8px;}

.wp-pagenavi {display:block; height:30px; margin-bottom:18px; text-shadow: #000 1px 1px 1px;} .wp-pagenavi span, .wp-pagenavi a {display:block; height:20px; padding:3px 8px 0 8px; text-align:center; float:left; background:#171612; margin-left:2px;}

.wp-pagenavi a:visited, .wp-pgenavi a:active {display:block; height:20px; padding:3px 8px 0 8px; text-align:center; float:left; background:#171612; margin-left:2px;} .wp-pagenavi .current, .wp-pagenavi a:hover {background:#000; color:#fff;}


</style>


</head>


<body>






<div id="footerlinks">

<ul id="footernav"> <li><a href="http://www.columbia.edu/">Columbia University</a> &raquo;</li> <li><a href="http://orion.bme.columbia.edu/hess/">Hess Lab</a> &raquo;</li> <!-- <li><a href="#">Articles</a> &raquo;</li> <li><a href="#">Contact</a> &raquo;</li> --> <li><a style="float: right;" href="http://www.twitter.com/KinesinKings" target="_blank">

 <img src="https://twitter.com/images/resources/twitter-bird-light-bgs.png" border="0" width="30" height="30" alt="Twitter" />

</a></li> </ul> </div> <div id="bottom">


 <p style="height:36px;">

<a><img src ="http://openwetware.org/images/5/54/CULogo.png" width="181" height="35" alt="Columbia" style ="float:right; margin-right:-45px;"/></a></p>


</div> </div> </body>

</html>