Bitan:SATA conjugation to Aβ

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<head> <meta name=Title content="Procedure for SATA-A&#946;40 conjugation"> <meta name=Keywords content=""> <meta http-equiv=Content-Type content="text/html; charset=macintosh"> <meta name=ProgId content=Word.Document> <meta name=Generator content="Microsoft Word 11"> <meta name=Originator content="Microsoft Word 11"> <link rel=File-List href="Procedure%20for%20SATA_files/filelist.xml"> <title>Procedure for SATA-A&#946;40 conjugation</title> <!--[if gte mso 9]><xml>

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<div class=Section1>

<table border=1 cellspacing=0 cellpadding=0 style='border-collapse:collapse;

border:none;mso-border-alt:solid windowtext .5pt;mso-padding-alt:0cm 5.4pt 0cm 5.4pt'>
<tr>
 <td width=730 valign=top style='width:729.55pt;border:solid windowtext .5pt;
 background:#FFCC99;mso-shading:windowtext;mso-pattern:solid #FFCC99;
 padding:0cm 5.4pt 0cm 5.4pt'>
 <p class=MsoNormal align=center style='text-align:center'><span
 style='font-size:16.0pt;font-family:Calibri'><b>Procedure for <a
 href="http://www.piercenet.com/Products/Browse.cfm?fldID=81058681-4C92-45CD-93AA-07E6B4C7F586&amp;WT.mc_id=keyname">SATA</a>-A&#946;40
 conjugation<o:p></o:p></b></span></p>
 <p class=MsoNormal align=center style='text-align:center'><span
 style='font-size:16.0pt;font-family:Calibri'><b>Small-scale prepartion<o:p></o:p></b></span></p>
 <ol style='margin-top:0cm' start=1 type=1>
  <li class=MsoNormal style='mso-list:l4 level1 lfo1;tab-stops:list 36.0pt'><span
      style='font-size:14.0pt;font-family:Calibri'>Weigh out 10&#8722;12 mg of
      peptide-bound resin. This resin has a Wang substitution of 0.27 mmol
      peptide/g resin. This amount will give 2.7&#8722;3.2 &#956;mol
      A&#946;40.<o:p></o:p></span></li>
  <li class=MsoNormal style='mso-list:l4 level1 lfo1;tab-stops:list 36.0pt'><span
      style='font-size:14.0pt;font-family:Calibri'>Swell the resin in 0.5 mL
      DMF (40 min) followed by DCM (0.5 mL, 40 min) and DMF (0.5 mL, 40 min)
      while shaken on the platform shaker/rocker.<o:p></o:p></span></li>
  <li class=MsoNormal style='mso-list:l4 level1 lfo1;tab-stops:list 36.0pt'><span
      style='font-size:14.0pt;font-family:Calibri'>Perform the Kaiser test<o:p></o:p></span></li>
 </ol>
 <p class=MsoNormal style='margin-left:72.0pt;text-indent:-18.0pt;mso-list:
 l4 level2 lfo1;tab-stops:list 72.0pt'><![if !supportLists]><span
 style='font-size:14.0pt;font-family:Calibri'>a.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
 </span></span><![endif]><span style='font-size:14.0pt;font-family:Calibri'>Take
 some resin out<o:p></o:p></span></p>
 <p class=MsoNormal style='margin-left:72.0pt;text-indent:-18.0pt;mso-list:
 l4 level2 lfo1;tab-stops:list 72.0pt'><![if !supportLists]><span
 style='font-size:14.0pt;font-family:Calibri'>b.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
 </span></span><![endif]><span style='font-size:14.0pt;font-family:Calibri'>Add
 the Kaiser reagents (2 drops or 15&#8722;20 &#956;L)<o:p></o:p></span></p>
 <p class=MsoNormal style='margin-left:72.0pt;text-indent:-18.0pt;mso-list:
 l4 level2 lfo1;tab-stops:list 72.0pt'><![if !supportLists]><span
 style='font-size:14.0pt;font-family:Calibri'>c.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
 </span></span><![endif]><span style='font-size:14.0pt;font-family:Calibri'>Mix
 well by vortex.<o:p></o:p></span></p>
 <p class=MsoNormal style='margin-left:72.0pt;text-indent:-18.0pt;mso-list:
 l4 level2 lfo1;tab-stops:list 72.0pt'><![if !supportLists]><span
 style='font-size:14.0pt;font-family:Calibri'>d.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
 </span></span><![endif]><span style='font-size:14.0pt;font-family:Calibri'>Heat
 at 100&#8722;120 °C for 4&#8722;6 min<o:p></o:p></span></p>
 <p class=MsoNormal style='margin-left:72.0pt;text-indent:-18.0pt;mso-list:
 l4 level2 lfo1;tab-stops:list 72.0pt'><![if !supportLists]><span
 style='font-size:14.0pt;font-family:Calibri'>e.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
 </span></span><![endif]><span style='font-size:14.0pt;font-family:Calibri'>Assess
 the color change. Blue color suggests presence of free amines.<o:p></o:p></span></p>
 <ol style='margin-top:0cm' start=4 type=1>
  <li class=MsoNormal style='mso-list:l4 level1 lfo1;tab-stops:list 36.0pt'><span
      style='font-size:14.0pt;font-family:Calibri'>If free amines are present,
      there is no need for deprotection.<o:p></o:p></span></li>
  <li class=MsoNormal style='mso-list:l4 level1 lfo1;tab-stops:list 36.0pt'><span
      style='font-size:14.0pt;font-family:Calibri'>Calculate amount of SATA,
      HBTU and DIPEA. Need to use 10-fold excess to A&#946;.<o:p></o:p></span></li>
 </ol>
 <p class=MsoNormal style='margin-left:72.0pt;text-indent:-18.0pt;mso-list:
 l4 level2 lfo1;tab-stops:list 72.0pt'><![if !supportLists]><span
 style='font-size:14.0pt;font-family:Calibri'>a.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
 </span></span><![endif]><span style='font-size:14.0pt;font-family:Calibri'>Mole
 of SATA needed=27 &#956;mol; SATA FW=231.23 g/mol; mass of SATA needed=27&times;10<sup>&#8722;6</sup>
 mol&times;231.23 = 6.24&times;13<sup>&#8722;3</sup> g =6.2 mg <o:p></o:p></span></p>
 <p class=MsoNormal style='margin-left:72.0pt;text-indent:-18.0pt;mso-list:
 l4 level2 lfo1;tab-stops:list 72.0pt'><![if !supportLists]><span
 style='font-size:14.0pt;font-family:Calibri'>b.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
 </span></span><![endif]><span style='font-size:14.0pt;font-family:Calibri'>Mole
 of HBTU needed=27 &#956;mol; HBTU FW=379.3 g/mol; mass of HBTU
 needed=27&times;10<sup>&#8722;6</sup> mol&times;379.3 g/mol=10.24 mg<o:p></o:p></span></p>
 <p class=MsoNormal style='margin-left:72.0pt;text-indent:-18.0pt;mso-list:
 l4 level2 lfo1;tab-stops:list 72.0pt'><![if !supportLists]><span
 style='font-size:14.0pt;font-family:Calibri'>c.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
 </span></span><![endif]><span style='font-size:14.0pt;font-family:Calibri'>Mole
 of DIPEA needed is 27 &#956;mol. DPEA &#961;=0.742 g/mL; FW=129.24 g/mol.
 Concentration of stock=0.742 g/mL÷129.24 g/mol=5.7&times;10<sup>&#8722;3</sup>
 mol/mL= 5.7 M. Volume of DIPEA needed= 27&times;10<sup>&#8722;6</sup> mol÷5.7
 mol/L=4.7 &#956;L.<o:p></o:p></span></p>
 <ol style='margin-top:0cm' start=6 type=1>
  <li class=MsoNormal style='mso-list:l4 level1 lfo1;tab-stops:list 36.0pt'><span
      style='font-size:14.0pt;font-family:Calibri'>Mix SATA, HBTU and DIPEA
      together and check the pH. <o:p></o:p></span></li>
  <li class=MsoNormal style='mso-list:l4 level1 lfo1;tab-stops:list 36.0pt'><span
      style='font-size:14.0pt;font-family:Calibri'>Add to resin and incubate
      in the CEM Microwave Discover running the appropriate coupling method.
      Run the coupling method once more.<o:p></o:p></span></li>
  <li class=MsoNormal style='mso-list:l4 level1 lfo1;tab-stops:list 36.0pt'><span
      style='font-size:14.0pt;font-family:Calibri'>Wash the resin profusely
      with DMF.<o:p></o:p></span></li>
  <li class=MsoNormal style='mso-list:l4 level1 lfo1;tab-stops:list 36.0pt'><span
      style='font-size:14.0pt;font-family:Calibri'>Wash the resin profusely
      with DCM.<o:p></o:p></span></li>
  <li class=MsoNormal style='mso-list:l4 level1 lfo1;tab-stops:list 36.0pt'><span
      style='font-size:14.0pt;font-family:Calibri'>Keep an aliquot for test
      cleavage.<o:p></o:p></span></li>
  <li class=MsoNormal style='mso-list:l4 level1 lfo1;tab-stops:list 36.0pt'><span
      style='font-size:14.0pt;font-family:Calibri'>Store the beads at
      &#8722;20°C or follow on to cleave the products using the CEM discover.<o:p></o:p></span></li>
 </ol>
 <p class=MsoNormal><span style='font-size:14.0pt;font-family:Calibri'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></span></p>
 <span style='font-size:16.0pt;font-family:Calibri;mso-ansi-language:EN-US;
 mso-fareast-language:EN-US'><b><br clear=ALL style='page-break-before:always'>
 </b></span>
 <p class=MsoNormal align=center style='text-align:center'><span
 style='font-size:16.0pt;font-family:Calibri'><b>Procedure for <a
 href="http://www.piercenet.com/Products/Browse.cfm?fldID=81058681-4C92-45CD-93AA-07E6B4C7F586&amp;WT.mc_id=keyname">SATA</a>-A&#946;40
 conjugation<o:p></o:p></b></span></p>
 <p class=MsoNormal align=center style='text-align:center'><span
 style='font-size:16.0pt;font-family:Calibri'><b>Large-scale prepartion<o:p></o:p></b></span></p>
 <ol style='margin-top:0cm' start=1 type=1>
  <li class=MsoNormal style='mso-list:l2 level1 lfo4;tab-stops:list 36.0pt'><span
      style='font-size:14.0pt;font-family:Calibri'>Weigh out 100 mg of
      peptide-bound resin. This resin has a Wang substitution of 0.27 mmol
      peptide/g resin. This amount will give ~27 &#956;mol A&#946;40. <o:p></o:p></span></li>
  <li class=MsoNormal style='mso-list:l2 level1 lfo4;tab-stops:list 36.0pt'><span
      style='font-size:14.0pt;font-family:Calibri'>Swell the resin in
      1&#8211;2 mL DMF (40 min) followed by DCM (1&#8211;2 mL, 40 min) and DMF
      (1&#8211;2 mL, 40 min) while shaken on the platform shaker/rocker.<o:p></o:p></span></li>
  <li class=MsoNormal style='mso-list:l2 level1 lfo4;tab-stops:list 36.0pt'><span
      style='font-size:14.0pt;font-family:Calibri'>Perform the Kaiser test<o:p></o:p></span></li>
 </ol>
 <p class=MsoNormal style='margin-left:72.0pt;text-indent:-18.0pt;mso-list:
 l2 level2 lfo4;tab-stops:list 72.0pt'><![if !supportLists]><span
 style='font-size:14.0pt;font-family:Calibri'>a.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
 </span></span><![endif]><span style='font-size:14.0pt;font-family:Calibri'>Take
 some resin out<o:p></o:p></span></p>
 <p class=MsoNormal style='margin-left:72.0pt;text-indent:-18.0pt;mso-list:
 l2 level2 lfo4;tab-stops:list 72.0pt'><![if !supportLists]><span
 style='font-size:14.0pt;font-family:Calibri'>b.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
 </span></span><![endif]><span style='font-size:14.0pt;font-family:Calibri'>Add
 the Kaiser reagents (2 drops or 15&#8722;20 &#956;L)<o:p></o:p></span></p>
 <p class=MsoNormal style='margin-left:72.0pt;text-indent:-18.0pt;mso-list:
 l2 level2 lfo4;tab-stops:list 72.0pt'><![if !supportLists]><span
 style='font-size:14.0pt;font-family:Calibri'>c.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
 </span></span><![endif]><span style='font-size:14.0pt;font-family:Calibri'>Mix
 well by vortex.<o:p></o:p></span></p>
 <p class=MsoNormal style='margin-left:72.0pt;text-indent:-18.0pt;mso-list:
 l2 level2 lfo4;tab-stops:list 72.0pt'><![if !supportLists]><span
 style='font-size:14.0pt;font-family:Calibri'>d.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
 </span></span><![endif]><span style='font-size:14.0pt;font-family:Calibri'>Heat
 at 100&#8722;120 °C for 4&#8722;6 min<o:p></o:p></span></p>
 <p class=MsoNormal style='margin-left:72.0pt;text-indent:-18.0pt;mso-list:
 l2 level2 lfo4;tab-stops:list 72.0pt'><![if !supportLists]><span
 style='font-size:14.0pt;font-family:Calibri'>e.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
 </span></span><![endif]><span style='font-size:14.0pt;font-family:Calibri'>Assess
 the color change. Blue color suggests presence of free amines.<o:p></o:p></span></p>
 <ol style='margin-top:0cm' start=4 type=1>
  <li class=MsoNormal style='mso-list:l2 level1 lfo4;tab-stops:list 36.0pt'><span
      style='font-size:14.0pt;font-family:Calibri'>If free amino group is
      present there is not need for deprotection.<o:p></o:p></span></li>
  <li class=MsoNormal style='mso-list:l2 level1 lfo4;tab-stops:list 36.0pt'><span
      style='font-size:14.0pt;font-family:Calibri'>Calculate amount of SATA,
      HBTU and DIPEA. Need to use <b>5-fold</b></span><span style='font-size:
      14.0pt;font-family:Calibri'> excess to A&#946;.<o:p></o:p></span></li>
 </ol>
 <p class=MsoNormal style='margin-left:72.0pt;text-indent:-18.0pt;mso-list:
 l2 level2 lfo4;tab-stops:list 72.0pt'><![if !supportLists]><span
 style='font-size:14.0pt;font-family:Calibri'>a.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
 </span></span><![endif]><span style='font-size:14.0pt;font-family:Calibri'>Mole
 of SATA needed=135 &#956;mol; SATA FW=231.23 g/mol; mass of SATA
 needed=135&times;10<sup>&#8722;6</sup> mol&times;231.23 = 3.12&times;13<sup>&#8722;2</sup>
 g =31.2 mg<o:p></o:p></span></p>
 <p class=MsoNormal style='margin-left:72.0pt;text-indent:-18.0pt;mso-list:
 l2 level2 lfo4;tab-stops:list 72.0pt'><![if !supportLists]><span
 style='font-size:14.0pt;font-family:Calibri'>b.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
 </span></span><![endif]><span style='font-size:14.0pt;font-family:Calibri'>Mole
 of HBTU needed=135 &#956;mol; HBTU FW=379.3 g/mol; mass of HBTU
 needed=135&times;10<sup>&#8722;6</sup> mol&times;379.3 g/mol=51.2 mg<o:p></o:p></span></p>
 <p class=MsoNormal style='margin-left:72.0pt;text-indent:-18.0pt;mso-list:
 l2 level2 lfo4;tab-stops:list 72.0pt'><![if !supportLists]><span
 style='font-size:14.0pt;font-family:Calibri'>c.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
 </span></span><![endif]><span style='font-size:14.0pt;font-family:Calibri'>Mole
 of DIPEA needed is 135 &#956;mol. DPEA &#961;=0.742 g/mL; FW=129.24 g/mol.
 Concentration of stock=0.742 g/mL÷129.24 g/mol=5.7&times;10<sup>&#8722;3</sup>
 mol/mL= 5.7 M. Volume of DIPEA needed= 135&times;10<sup>&#8722;6</sup>
 mol÷5.7 mol/L=23.5 &#956;L.<o:p></o:p></span></p>
 <ol style='margin-top:0cm' start=6 type=1>
  <li class=MsoNormal style='mso-list:l2 level1 lfo4;tab-stops:list 36.0pt'><span
      style='font-size:14.0pt;font-family:Calibri'>Mix SATA, HBTU and DIPEA
      together and check the pH.<o:p></o:p></span></li>
  <li class=MsoNormal style='mso-list:l2 level1 lfo4;tab-stops:list 36.0pt'><span
      style='font-size:14.0pt;font-family:Calibri'>Add to resin and incubate
      in the CEM Microwave Discover running the appropriate coupling method.
      Run the coupling method once more.<o:p></o:p></span></li>
  <li class=MsoNormal style='mso-list:l2 level1 lfo4;tab-stops:list 36.0pt'><span
      style='font-size:14.0pt;font-family:Calibri'>Wash the resin profusely
      with DMF.<o:p></o:p></span></li>
  <li class=MsoNormal style='mso-list:l2 level1 lfo4;tab-stops:list 36.0pt'><span
      style='font-size:14.0pt;font-family:Calibri'>Wash the resin profusely
      with DCM.<o:p></o:p></span></li>
  <li class=MsoNormal style='mso-list:l2 level1 lfo4;tab-stops:list 36.0pt'><span
      style='font-size:14.0pt;font-family:Calibri'>Keep an aliquot for test
      cleavage.<o:p></o:p></span></li>
  <li class=MsoNormal style='mso-list:l2 level1 lfo4;tab-stops:list 36.0pt'><span
      style='font-size:14.0pt;font-family:Calibri'>Store the beads at
      &#8722;20°C or follow on to cleave the products using the CEM discover.<o:p></o:p></span></li>
  <li class=MsoNormal style='mso-list:l2 level1 lfo4;tab-stops:list 36.0pt'><span
      style='font-size:14.0pt;font-family:Calibri'>Cleave with cleavage
      solution 825 &#956;L TFA + 50 &#956;L H<sub>2</sub>O + 50 &#956;L
      thioanisol + 25 &#956;L EDT. For test-cleavage, use 30&#8211;60 &#956;L
      of this solution. Incubate shakingly for 3&#8211;4 hours.<o:p></o:p></span></li>
  <li class=MsoNormal style='mso-list:l2 level1 lfo4;tab-stops:list 36.0pt'><span
      style='font-size:14.0pt;font-family:Calibri'>Add 5 &#956;L of this
      solution to 200 &#956;L of 50% methanol in water and inject
      150&#8211;170 &#956;L into LC-MS.<o:p></o:p></span></li>
 </ol>
 <p class=MsoNormal align=center style='text-align:center'><span
 style='font-size:14.0pt;font-family:Calibri'><a
 href="http://openwetware.org/wiki/Bitan:todo">Back to To-Do List</a></span><span
 style='font-size:16.0pt;font-family:Calibri'><b><o:p></o:p></b></span></p>
 </td>
</tr>

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