Biomod/2011/TUM/TNT/LabbookA/Purification of origamis by size exclusion filtration

From OpenWetWare
Jump to: navigation, search

<html>

  <style>
  1. column-one { display:none; width:0px;}

.container{background-color: #f5f5f5; margin-top:50px} .OWWNBcpCurrentDateFilled {display: none;}

  1. content { width: 0px; margin: 0 auto auto 0; padding: 1em 1em 1em 1em; align: center;}
  1. column-content {width: 0px; float: left; margin: 0 0 0 0;padding: 0;}

.firstHeading {display:none; width:0px;}

  1. globalWrapper{ width:1280px;}

div.tright { border-bottom-width: 0em; border-left-width: 0px; border-right-width: 0px; border-top-width: 0em; clear: right; float: right;

}

div.tleft { border-bottom-width: 0em; border-left-width: 0px; border-right-width: em; border-top-width: 0em; clear: left; float: left; margin-right: 0.5em;

}

div.tnone { border-bottom-width: 0em; border-left-width: 0px; border-right-width: em; border-top-width: 0em; }

  1. column-one {display:none; width:0px;background-color: #ffffff;}
  1. content{ margin: 0 0 0 0; padding: 1em 1em 1em 1em; position: center; width: 800px;background-color: #f5f5f5; }

.container{ width: 800px; margin: auto; background-color: #ffffff; text-align:justify; font-family: helvetica, arial, sans-serif; color:#555555; margin-top:25px; }

  1. bodyContent{ width: 1267px; align: center; background-color: #ffffff;}
  1. column-content{width: 1280px;background-color: #f5f5f5;}

.firstHeading { display:none;width:0px;background-color: #ffffff;}

  1. header{position: center; width: 800px;background-color: #ffffff;}
  1. footer{position: center;}

<style type="text/css"> body {

   font-family: helvetica, arial, sans-serif;
   color: black;

background:#f5f5f5;

 }
ul#topnav {

margin: 0 0 0 0px ; padding: 0 0 0 0px; float: left; width: 800px; list-style: none; position: relative;

       font-family: helvetica, arial, sans-serif;

font-size: 12px; background: #f5f5f5;

       height: 5px;

} ul#topnav li { float: left; margin: 0; padding: 0; border-right: 1px solid #ffffff; /*--Divider for each parent level links--*/ } ul#topnav li a { padding: 11px 15px; display: block; color: #333333; text-decoration: none; } ul#topnav li:hover { background: #f5f5f5;} /*--Notice the hover color is on the list item itself, not on the link. This is so it can stay highlighted even when hovering over the subnav--*/

ul#topnav li span { float: left; padding: 15px 0; position: absolute; left: 0; top:30px; display: inline; /*--Hide by default--*/ width: 800px; background: #cccccc; color: #333333; /*--Bottom right rounded corner--*/ -moz-border-radius-bottomright: 5px; -khtml-border-radius-bottomright: 5px; -webkit-border-bottom-right-radius: 5px; /*--Bottom left rounded corner--*/ -moz-border-radius-bottomleft: 5px; -khtml-border-radius-bottomleft: 5px; -webkit-border-bottom-left-radius: 5px; } ul#topnav li:hover span { display: block; } /*--Show subnav on hover--*/ ul#topnav li span a { display: inline;} /*--Since we declared a link style on the parent list link, we will correct it back to its original state--*/ ul#topnav li span a:hover {text-decoration: underline;}


  1. abstractandlinks{

display:block; width:800px; background-color:#f5f5f5; text-align:justify;font-family: helvetica, arial, sans-serif; color:#555555; margin-top:0px; }

  1. innerBox

{ display: block; padding: 5px; }

  1. abstract{

font-family: helvetica, arial, sans-serif; width: 490px; margin-top: 20px; padding:0px 5px; float: left;background-color:#f5f5f5; ; }

  1. links{

font-family: helvetica, arial, sans-serif; width: 290px; margin-top: 20px; padding:0px 5px; float: left; background-color:#f5f5f5;; } .clear { clear:both; }

  1. tour{

font-family: helvetica, arial, sans-serif; width: 790px; padding:0px 5px; margin-top: 1em, margin-bottom: 1em; float: left;background:#f5f5f5; font-size: 2em; }

  1. struktur{

font-family: helvetica, arial, sans-serif; width: 790px; padding:0px 5px; float: left;background:#f5f5f5; }

  1. toctitle {align: left; text-align: left; }
  2. table.toc {background-color:"#5f5f5f";align: left; text-align: left;}
  3. toc #toctitle, .toc #toctitle, #toc .toctitle, .toc .toctitle {

text-align: left;

}
  1. toc, .toc, .mw-warning {

background-color: #f5f5f5; border-bottom-color: rgb(170, 170, 170); border-bottom-style: solid; border-bottom-width: 0px; border-left-color: rgb(170, 170, 170); border-left-style: solid; border-left-width: 0px; border-right-color: rgb(170, 170, 170); border-right-style: solid; border-right-width: 0px; border-top-color: rgb(170, 170, 170); border-top-style: solid; border-top-width: 0px; font-size: 105%; padding-bottom: 5px; padding-left: 5px; padding-right: 5px; padding-top: 5px; align: left; text-align: left; display: left

}


</style>

<head>

<script type="text/javascript">


$(document).ready(function() {

$("ul#topnav li").hover(function() { //Hover over event on list item $(this).css({ 'background' : '#cccccc'}); //Add background color and image on hovered list item $(this).find("span").show(); //Show the subnav

} ,

$("ul#topnav li").oneclick(function() { //Hover over event on list item $(this).css({ 'background' : '#cccccc'; 'display' : 'inline'}); //Add background color and image on hovered list item $(this).find("span").show(); //Show the subnav

} ,

function() { //on hover out...


$(this).css({ 'background' : 'none'}); //Ditch the background

//$(this).find("span").hide(); //Hide the subnav });

}); </script>


<title>

       TUM NanU - Home   

</title> </head>

<body background-color="#F0F0F0"> <!-- Header--> <div class="container">

   <div id="header"> <img src="http://openwetware.org/images/5/5c/Header2.png" /> </div> 
   

<!--Menue--> <ul id="topnav">

   <li><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Home">Home</a></li>
   <li><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Project">Project</a>        
      </li>
   <li><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results">Results & Discussion</a>        
   </li>
   <li><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Methods">Methods</a></li>
      
   <li><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Team">Team</a></li>
<li style="background-color: #cccccc"><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA"><b>Labbook</b></a><!--Subnav Starts Here-->
       <span>
           <a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA">Main Page</a>||
           <a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Structure_page">Structure</a>|
           <a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/list_TEM_pictures">TEM</a>|
           <a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/2011/TIRF">Fluorescence Microscopy</a>|
           <a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Glossary">Glossary</a>
        </span>
   </li>
   <li><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Extras">Extras</a>
       
  </li>

</ul> <p> <br> </p> <p> <br> </p> <p> <br> </p> </html>

<html>

<html> <head> <script type= "text/javascript">/*<![CDATA[*/ var skin = "monobook"; var stylepath = "/skins"; var wgArticlePath = "/wiki/$1"; var wgScriptPath = ""; var wgScript = "/index.php"; var wgVariantArticlePath = false; var wgActionPaths = []; var wgServer = "http://openwetware.org"; var wgCanonicalNamespace = ""; var wgCanonicalSpecialPageName = false; var wgNamespaceNumber = 0; var wgPageName = "Biomod/2011/TUM/TNT/LabbookA/2011"; var wgTitle = "Biomod/2011/TUM/TNT/LabbookA/2011 var wgAction = "view"; var wgArticleId = "103708"; var wgIsArticle = true; var wgUserName = "Alexander Hipp"; var wgUserGroups = ["*", "user", "autoconfirmed"]; var wgUserLanguage = "en"; var wgContentLanguage = "en"; var wgBreakFrames = false; var wgCurRevisionId = "543703"; var wgVersion = "1.13.2"; var wgEnableAPI = true; var wgEnableWriteAPI = false; var wgMWSuggestTemplate = "http://openwetware.org/api.php?action=opensearch\x26search={searchTerms}\x26namespace={namespaces}"; var wgDBname = "owwdb"; var wgSearchNamespaces = [0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 100, 101, 110, 111]; var wgMWSuggestMessages = ["with suggestions", "no suggestions"]; var wgRestrictionEdit = []; var wgRestrictionMove = []; var wgAjaxWatch = {"watchMsg": "Watch", "unwatchMsg": "Unwatch", "watchingMsg": "Watching...", "unwatchingMsg": "Unwatching..."}; /*]]>*/</script>

<script type="text/javascript" src="/skins/common/wikibits.js?164"><!-- wikibits js --></script> <!-- Head Scripts --> <script type="text/javascript" src="/js/calendar.js"></script> <script type="text/javascript"> function lnCalendar(){

 var calm1 = new CalendarPopup('m1');
 var wikiPage = 'Biomod/2011/TUM/TNT/LabbookA';
 var redirPage = '/wiki/Special:Redir/'+wikiPage;
 var fullDates = new Array('08/11/2011','08/17/2011','08/23/2011','08/24/2011','08/25/2011','08/29/2011','08/30/2011','09/01/2011','09/02/2011','09/06/2011','09/12/2011','09/13/2011','09/14/2011','09/15/2011','09/16/2011','09/19/2011','09/20/2011','09/21/2011','09/27/2011','09/28/2011','09/29/2011','09/30/2011','10/05/2011','10/06/2011','10/07/2011','10/10/2011','10/12/2011','10/13/2011','10/14/2011','10/17/2011','10/18/2011','10/19/2011','10/20/2011','10/21/2011','10/26/2011','10/27/2011','10/28/2011','10/30/2011','10/31/2011');
 calm1.setDateFormat('yyyy/MM/dd');
 calm1.setCssPrefix('OWWNB');
 calm1.setSingleMonth('8');
 calm1.setYear('2011');
 calm1.setReadOnly('N');
 calm1.setTodayText('');
 calm1.setUrlPrefix(redirPage);
 for(i = 0; i < fullDates.length; i++)calm1.addFilledDates(fullDates[i]);
 calm1.showCalendar('m1');
 var calm2 = new CalendarPopup('m2');

('08/11/2011','08/12/2011','08/16/2011','08/17/2011','08/23/2011','08/24/2011','08/25/2011','08/29/2011','08/30/2011','09/01/2011','09/02/2011','09/06/2011','09/12/2011','09/13/2011','09/14/2011','09/15/2011','09/16/2011','09/19/2011','09/20/2011','09/21/2011','09/27/2011','09/28/2011','09/29/2011','09/30/2011','10/05/2011','10/06/2011','10/07/2011','10/10/2011','10/12/2011','10/13/2011','10/14/2011','10/17/2011','10/18/2011','10/19/2011','10/20/2011','10/21/2011','10/26/2011','10/27/2011','10/28/2011','10/30/2011','10/31/2011');

 calm2.setDateFormat('yyyy/MM/dd');
 calm2.setCssPrefix('OWWNB');
 calm2.setSingleMonth('9');
 calm2.setYear('2011');
 calm2.setReadOnly('N');
 calm2.setTodayText('');
 calm2.setUrlPrefix(redirPage);
 for(i = 0; i < fullDates.length; i++)calm2.addFilledDates(fullDates[i]);
 calm2.showCalendar('m2');
 var calm3 = new CalendarPopup('m3');

('08/11/2011','08/12/2011','08/16/2011','08/17/2011','08/23/2011','08/24/2011','08/25/2011','08/29/2011','08/30/2011','09/01/2011','09/02/2011','09/06/2011','09/12/2011','09/13/2011','09/14/2011','09/15/2011','09/16/2011','09/19/2011','09/20/2011','09/21/2011','09/27/2011','09/28/2011','09/29/2011','09/30/2011','10/05/2011','10/06/2011','10/07/2011','10/10/2011','10/12/2011','10/13/2011','10/14/2011','10/17/2011','10/18/2011','10/19/2011','10/20/2011','10/21/2011','10/26/2011','10/27/2011','10/28/2011','10/30/2011','10/31/2011');

 calm3.setDateFormat('yyyy/MM/dd');
 calm3.setCssPrefix('OWWNB');
 calm3.setSingleMonth('10');
 calm3.setYear('2011');
 calm3.setReadOnly('N');
 calm3.setTodayText('');
 calm3.setUrlPrefix(redirPage);
 for(i = 0; i < fullDates.length; i++)calm3.addFilledDates(fullDates[i]);
 calm3.showCalendar('m3');

} </script> <script type="text/javascript">addOnloadHook( lnCalendar )</script> <script type="text/javascript">function CalendarPageConfirmCreate(date, url){var answer = confirm("Create entry for "+date+"?");if (answer){window.location = url;}}</script> <script type="text/javascript" src="/skins/common/ajax.js?164"></script> <script type="text/javascript" src="/ext/AjaxShowEditors/AjaxShowEditors.js"></script> <script type="text/javascript" src="/skins/common/ajaxwatch.js?164"></script> <script type="text/javascript" src="/skins/common/mwsuggest.js?164"></script> <script type="text/javascript" src="/index.php?title=-&amp;action=raw&amp;smaxage=0&amp;gen=js&amp;useskin=monobook"><!-- site js --></script>


<style type="text/css">/*<![CDATA[*/ @import "/index.php?title=MediaWiki:Common.css&amp;usemsgcache=yes&amp;action=raw&amp;ctype=text/css&amp;smaxage=18000"; @import "/index.php?title=MediaWiki:Monobook.css&amp;usemsgcache=yes&amp;action=raw&amp;ctype=text/css&amp;smaxage=18000"; @import "/index.php?title=-&amp;action=raw&amp;gen=css&amp;maxage=18000&amp;smaxage=0&amp;ts=20111012100110&amp;useskin=monobook"; @import "/index.php?title=User:Alexander_Hipp/monobook.css&amp;action=raw&amp;ctype=text/css"; /*]]>*/</style> <script type="text/javascript" src="/index.php?title=User:Alexander_Hipp/monobook.js&amp;action=raw&amp;ctype=text/javascript"></script> </head>

</html>

Purification of origamis by size exclusion filtration

The first folded origamis were purified by agarose gel electrophoresis. This method is well characterized, but takes several hours and a huge amount of origami is lost in the gel. Since our structure folds quite properly and almost no misfolded origamis can be seen, such a thorough purification is not necessary in our case. We just need to get rid of the unused staples. Therefore, on the 6th September we tested purification with Amicon size exclusion filters for the first time. The molecular weight cutoff is 100 kDa, so staples are filtered out, while origamis pass the filter.
First experiments were quite successful, insofar as the loss due to the filter was considerably smaller as that due to electrophoresis. Later, a PhD student of our lab tested varying conditions to optimize purity and minimize loss of origamis. All filtrations after the 14th September were done according to the following protocol, which gave the best results (i.e. no superfluous staples, good yield) in Martys experiments:

  • apply 100 µl sample into an Amicon filter (100 kDa)
  • add 400 µl FOB20
  • spin down for 2 minutes at 14000 rcf at 4°C
  • repeat washing for three times
  • add 400 µl FOB20
  • spin down for 5 minutes at 14000 rcf at 4°C
  • turn the filter and spin down the purified sample into a new Epi for 2 minutes at 1000 rcf