Biomod/2014/Kashiwa/Protocol

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       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Design#2" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">The Receptor</span></a></li>
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   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Protocols" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">PROTOCOL</span></a>
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<body> <font face="Futura,Arial,Frutiger" font size="24px">PROTOCOL</font> <br> <br> <div class="CollapsibleBox" id="CBoxCover1">

  <div class="CollapsibleBoxTitle">
     <p class="cBoxButtons">
        <a href="#" onclick="HideCBox('CBoxBody1');  return false;" title="折りたたみ／復元"><font color="white" font size="2">[show/hide]</font></a>
     </p>
     <p class="cBoxTitle">&nbsp; Contents：</p>
  </div>
  <div class="CollapsibleBoxBody" id="CBoxBody1">
     <ul style="list-style:none;">
        <li> <a href=#1>1. Polymerization </a>
        <ul style="list-style:none;">
           <li> <a href="#1-1">1-1. Making the Monomer </a></li>
           <li> <a href="#1-2">1-2. Controlling polymerization </a></li>
           <li> <a href="#1-3">1-3. Putting the Monomer into a liposome </a></li>
           <li> <a href="#1-4">1-4. Polymerization </a>
           <ul style="list-style:none;">
              <li> <a href="#1-4-1">1-4-1. Polymerization in solution </a></li>
              <li> <a href="#1-4-2">1-4-2. Polymerization in a liposome </a></li>
           </ul>
           </li>
           <li> <a href="#1-5">1-5. Deformation of a liposome </a></li>
        </ul>
        </li>
        <li> <a href="#2">2. Receptor </a>
        <ul style="list-style:none">
           <li> <a href="#2-1">2-1. Making the Receptor </a></li>
           <li> <a href="#2-2">2-2. Penetration into a liposome </a></li>
           <li> <a href="#2-3">2-3. Dimerization
           <ul style="list-style:none">
              <li> <a href="#2-3-1">2-3-1. Dimerization in solution </a></li>
              <li> <a href="#2-3-2">2-3-2. Dimerization on a liposome </a></li>
           </ul>
           </li>
           <li> <a name="1"><a href="#2-4">2-4. Emission of polymerization initiator by dimerization </a></a>
           <ul style="list-style:none">
              <li> <a href="#2-4-1">2-4-1. Emission by dimerization in solution </a></li>
              <li> <a name="1-1"><a href="#2-4-2">2-4-2. Emission by dimerization on a liposome </a></a></li>
           </ul>
           </li>
        </ul>
     </ul>
  </div>

</div> <h1 class="title"><a name="contents"> 1. Polymerization</a></h1>

<p class="small">1-1. Making the Monomer</p> <table class="sample_01"> <tbody> <tr> <th>Reagents (f. 60 µL)</th> <th> </th> </tr> <tr> <td>Staple mix</td> <td>17.5 µL</td> </tr> <tr> <td>M 13 mp 18 ss</td> <td>18.3 µL</td> </tr> <tr> <td>TE¹</td> <td>18.2 µL</td> <tr> <td>10 × tile buffer²</td> <td> 6.0 µL</td> </tr> </tr> </tbody> </table>

<table class="sample_02"> <tbody> <tr> <th>²10 × tile buffer(f. 500 mL)</th> <th> </th> </tr> <tr> <td>Mg (OAc) <sub>2</sub> (f. 100 mM)</td> <td>10.73 g</td>

</tr> <tr> <td>1.0 M Tris-HCl (pH 7.5) (f. 200 mM)</td> <td> 100 mL</td> </tr> <tr> <td>0.5 M EDTA (f. 10 mM)</td> <td>10 mL</td> </tr> <tr> <td>MQ</td> <td>up to 500 mL</td> </tr> </tr> </tbody> </table>

<p>Procedure</p> <p>1) The reagents were mixed.</p> <p>2) The mixture was annealed at 55 °C for 3 hours.</p> <p>3) The mixture was analyzed by 1 % agarose gel electrophoresis.</p> <p>4) The gel was stained by EtBr for 30 minutes.</p> <p>5) The gel was taken photo by LAS - 4000.</p> <br>

<a name="1-2"> </a> <br><br><br><br> <p class="small">1-2. Controlling polymerization</p>

<a name="1-3"> </a> <br><br><br><br> <p class="small">1-3. Putting the Monomer into a liposome</p> <table class="sample_01"> <tbody> <tr> <th>Reagents (f.1420 µL)</th> <th> </th> </tr> <tr> <td>Lipid mix (f. 0.5 mM)¹</td> <td>400 µL</td> </tr> <tr> <td>Glucose (f. 200 mM)</td> <td>500 µL</td> </tr> <tr> <td>Emulsion mix²</td> <td>520 µL</td> </tr> </tbody> </table>

<table class="sample_02"> <tbody> <tr> <th>¹Lipid mix (f. 3 mM)</th> <th> </th> </tr> <tr> <td>POPC</td> <td>12 mg</td> </tr> <tr> <td>Paraffin</td> <td>5 mL</td> </tr> </tr> </tbody> </table>

<table class="sample_02"> <tbody> <tr> <th>²Emulsion mix</th> <th> </th> </tr> <tr> <td>Lipid mix (f. 0.5mM)</td> <td>500 µL</td> </tr> <tr> <td>Inner solution³</td> <td>10 µL</td> </tr> <tr> <td>The Monomer</td> <td>10 µL</td> </tr> </tr> </tbody> </table>

<table class="sample_02"> <tbody> <tr> <th>³Inner solution</th> <th> </th> </tr> <tr> <td>HEPES (f. 50 mM)</td> <td>250 µL</td> </tr> <tr> <td>Sucrose(f. 400 mM)</td> <td>250 µL</td> </tr> <tr> <td>Pyranine</td> <td>2 µL</td> </tr> </tr> </tbody> </table>

<p>Procedure</p> <p>1) Take glucose (500 µL) into a tube as outer solution.</p> <p>2) Lipid mix (0.5 mM) was added on the glucose solution carefully.</p> <p>3) Emulsion mix was added on the top and rapidly centrifuged at 4 °C, 1800 rpm for 10 minutes.</p> <p>4) Centrifuged again at 4 °C, 5000 rpm for 10 minutes.</p> <p>5) The upper layer was removed and GUVs were collected using pipetman.</p> <p>6) The liposome was stained by Nile red and observed with a confocal microscope.</p> <br>

<a name="1-4"> </a><br><br> <a name="1-4-1"> </a><br><br><br> <p class="small">1-4. Polymerization</p>

<p class="small">  1-4-1. Polymerization in solution</p> <table class="sample_01"> <tbody> <tr> <th>Reagents</th> <th> </th> </tr> <tr> <td>Purified Monomer A</td> <td>10 µL</td> </tr> <tr> <td>Purified Monomer B</td> <td>10 µL</td> </tr> </tbody> </table> <p>Procedure</p> <p>1) Monomers were made as shown in “Making DNA origami monomers”.</p> <p>2) Each Monomer solution was purified by spin column.</p> <p>3) The reagents was mixed.</p> <p>4) The mixture was incubated at room temperature for 24 hours.</p> <p>5) The mixture was analyzed by 1 % agarose gel electrophoresis (100 V, 80 min).</p> <p>6) The gel was stained by EtBr for 30 minutes.</p> <p>7) The gel was taken photo by LAS - 4000.</p>

<a name="1-4-2"> </a><br><br><br><br> <p class="small">  1-4-2. Polymerization in a liposome

<a name="2"> </a><br><br><br> <a name="2-1"> </a> <h1 class="title"><a name="contents"> 2. Receptor</a></h1>

<p class="small">2-1. Making the Receptor</p> <table class="sample_01"> <tbody> <tr> <th>Reagents (f. 40 µL)</th> <th> </th> </tr> <tr> <td>M 13 mp 18 ss</td> <td>18 µL</td> </tr> <tr> <td>Staple mix</td> <td>18 µL</td> </tr> <tr> <td>10 × OCK buffer¹</td> <td>4 µL</td> </tr> </body> </table>

<table class="sample_02"> <tbody> <tr> <th>¹10 × OCK buffer (f: 10 mL)</th> <th> </th> </tr> <tr> <td>1 M Tris (HCl pH 7.5) (f. 50 mM)</td> <td>500 µL</td> </tr> <tr> <td>0.5 M EDTA-Na (pH 8) (f. 10 mM)</td> <td>200 µL</td> </tr> <tr> <td>1 M MgCl<sub>2</sub> (f. 100 mM)</td> <td>1 mL</td> </tr> <tr> <td>5 M NaCl (f. 500 mM)</td> <td>100 µL</td> </tr> <tr> <td>MQ</td> <td>8.2 mL</td> </tr> </tbody> </table>

<p>Procedure</p> <p>1)the solutions were mixed.</p> <p>2) The mixture was annealed at 47.5 °C for 4 hours.</p> <p>3) The mixture was purified by spin column.</p> <p>4) The mixture was analyzed by 1% agarose gel electrophoresis (100V, 40 min).</p> <br>

<a name="2-2"> </a><br><br><br><br> <p class="small">2-2. Penetration into a liposome</p>

<p class="small">Ⅰ. Preparation of GUVｓ</p> <p>GUVs were made as shown in "Putting the Monomer into a liposome".</p>

<p class="small">Ⅱ. Preparation of LUVｓ</p> <br>

<p class="small">Ⅲ. Hybridization of cholesterol oligomer with the Receptor</p> <table class="sample_01"> <tbody> <tr> <th>Reagents (f. 7.5 µL)</th> <th> </th> </tr> <tr> <td>Purified Receptor (0.05 µM)</td> <td>6 µL</td> </tr> <tr> <td>Cholesterol oligomer (8 µM)</td> <td>1.5 µL</td> </tr> </tr> </tbody> </table>

<p>Procedure</p> <p> The reagents were mixed and incubated at room temperature for 60 minutes.</p>

<p class="small">Ⅳ. Flotation assay</p>

<table class="sample_01"> <tbody> <tr> <th>Reagents</th> <th> </th> </tr> <tr> <td>The Receptor</td> <td>100 µL</td> </tr> <tr> <td>Cholesterol hybridized Receptor</td> <td>100 µL</td> </tr> <tr> <td>Liposomes (? mg/mL LUVs)</td> <td>100 µL</td> </tr> <tr> <td>2.25 M Sucrose buffer¹</td> <td>500 µL</td> </tr> <tr> <td>1.6 M Sucrose buffer²</td> <td>900 µL</td> </tr> <tr> <td>150 mM KCl solution</td> <td>100 µL</td> </tr> <tr> <td>1 × Flotation buffer³</td> <td>600 µL</td> </tr> </tr> </tbody> </table>

<table class="sample_02"> <tbody> <tr> <th>¹2.25 M Sucrose buffer (f. ????)</th> <th> </th> </tr> <tr> <td>HEPES - KOH (pH 7.6) (f. 50 mM)</td> <td>?????</td> </tr> <tr> <td>KCl (f. 100 mM)</td> <td>?????</td> </tr> <tr> <td>MgCl<sub>2</sub> (f. 20 mM)</td> <td>?????</td> </tr> <tr> <td>Sucrose (f. 2.25 M)</td> <td>?????</td> </tr> </tr> </tbody> </table>

<table class="sample_02"> <tbody> <tr> <th>²1.6 M Sucrose buffer (f. ????)</th> <th> </th> </tr> <tr> <td>HEPES - KOH (pH 7.6) (f. 50 mM)</td> <td>?????</td> </tr> <tr> <td>KCl (f. 100 mM)</td> <td>?????</td> </tr> <tr> <td>MgCl<sub>2</sub> (f. 20 mM)</td> <td>?????</td> </tr> <tr> <td>Sucrose (f. 1.6 M)</td> <td>?????</td> </tr> </tr> </tbody> </table>

<table class="sample_02"> <tbody> <tr> <th>³1 × Flotation buffer (f. ?????)</th> <th> </th> </tr> <tr> <td>HEPES - KOH (pH 7.6) (f. 50 mM)</td> <td>?????</td> </tr> <tr> <td>KCl (f. 100 mM)</td> <td>?????</td> </tr> <tr> <td>MgCl<sub>2</sub> (f. 20 mM)</td> <td>?????</td> </tr> </tbody> </table>

<p>Procedure</p> <p>1) Each sample was mixed as shown below　(table1&sup4;).</p> <p>2) 225 µL of 1.6 M sucrose buffer was overlaid with 225 µL of sample mixture in centrifuge tubes (Beckman, cat#343778, 11 × 34 mm).</p> <p>3) Centrifuge for 16 minutes at 100 krpm at 4 ℃ using TLA 100.2 rotor (BECKMAN COULTER) with Ultracentrifuge (BECKMAN COULTER, Optima MAX-XP).</p> <p>4) 150 µL of supernatant was extracted from top to bottom for 3 times (Fraction 1 to 3) and the pellet was retrieved with 150 µL of 1 × Flotation buffer (Fraction 4).</p> <p>5) Fraction 1-4 of each sample were analyzed by 1 % agaraose gel electrophoresis (100V, 1 hour).</p> <p>6) The Intensity of fluorescence of NileRed (Liposomes) was measured with fluorescence spectrophotometer (JASCO, FP-6500) to investigate the existence of liposomes in each Fraction.</p> <p>7) The radiuses of liposome of each fraction were measured with DLS (Viscotek, 802 DLS).</p>

<table class="sample_04"> <caption>&sup4;Table 1. Breakdown of Samples</caption> <tbody> <tr> <th>sample No.</th> <td>1</td> <td>2</td> <td>3</td> <td>4</td> </tr> <tr> <th>Cholesterol hybridized Receptor</th> <td>50 µL</td> <td>50 µL</td> <td>—</td> <td>—</td> </tr> <tr> <th>Receptor</th> <td>—</td> <td>—</td> <td>50 µL</td> <td>50 µL</td> </tr> <tr> <th>Liposome</th> <td>50 µL</td> <td>—</td> <td>50 µL</td> <td>—</td> </tr> <tr> <th>150 mM aqueous KCl solution</th> <td>—</td> <td>50 µL</td> <td>—</td> <td>50 µL</td> </tr> <tr> <th>2.25 M Sucrose buffer</th> <td>125 µL</td> <td>125 µL</td> <td>125 µL</td> <td>125 µL</td> </tr> </tr> </tbody> </table> <br>

<p class="small">2-3. Dimerization</p>

<p class="small">  2-3-1. Dimerization in solution</p> <table class="sample_01"> <tbody> <tr> <th>Reagents</th> <th> </th> </tr> <tr> <td>Receptor E</td> <td> </td> </tr> <tr> <td>Receptor A</td> <td> </td> </tr> <tr> <td>Thrombin (1.09µM)</td> <td> </td> </tr> <tr> <td>Loading-dye</td> <td> </td> </tr> </tbody> </table> <p>Procedure</p> <p>1) Mix the solutions, and incubated 1 hour at room temperature.</p> <a name="2-3-2"> </a> <p>2) Analyzed by 1% agarose gel electrophoresis (100V, 80 min).</p>

<br> <p class="small">  2-3-2. Dimerization on a liposome</p>

<a name="2-4"> </a> <br><br> <a name="2-4-1"> </a> <br><br> <p class="small">2-4. Emission of polymerization initiator by dimerization</p>

<p class="small">  2-4-1. Emission by dimerization in solution</p>

<a name="2-4-2"> </a> <br> <p class="small">  2-4-2. Emission by dimerization on a liposome</p>

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Biomod/2014/Kashiwa/Protocol

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