Bitan:SATA conjugation to Aβ
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<head> <meta name=Title content="Procedure for SATA-Aβ40 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-Aβ40 conjugation</title> <!--[if gte mso 9]><xml>
<o:DocumentProperties> <o:Author>Farid Rahimi</o:Author> <o:Template>Normal</o:Template> <o:LastAuthor>Farid Rahimi</o:LastAuthor> <o:Revision>4</o:Revision> <o:LastPrinted>2008-01-17T18:24:00Z</o:LastPrinted> <o:Created>2009-12-02T21:06:00Z</o:Created> <o:LastSaved>2009-12-02T21:31:00Z</o:LastSaved> <o:Pages>1</o:Pages> <o:Words>617</o:Words> <o:Characters>2409</o:Characters> <o:Company>UCLA</o:Company> <o:Lines>160</o:Lines> <o:Paragraphs>160</o:Paragraphs> <o:CharactersWithSpaces>4323</o:CharactersWithSpaces> <o:Version>11.1282</o:Version> </o:DocumentProperties> <o:OfficeDocumentSettings> <o:AllowPNG/> </o:OfficeDocumentSettings>
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</head>
<body bgcolor=white lang=EN-US link=blue vlink=purple style='tab-interval:36.0pt'>
<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&WT.mc_id=keyname">SATA</a>-Aβ40 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 10−12 mg of
peptide-bound resin. This resin has a Wang substitution of 0.27 mmol
peptide/g resin. This amount will give 2.7−3.2 μmol
Aβ40.<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"'> </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"'> </span></span><![endif]><span style='font-size:14.0pt;font-family:Calibri'>Add the Kaiser reagents (2 drops or 15−20 μL)<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"'>
</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"'> </span></span><![endif]><span style='font-size:14.0pt;font-family:Calibri'>Heat at 100−120 °C for 4−6 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"'> </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 Aβ.<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"'>
</span></span><![endif]><span style='font-size:14.0pt;font-family:Calibri'>Mole
of SATA needed=27 μmol; SATA FW=231.23 g/mol; mass of SATA needed=27×10<sup>−6</sup>
mol×231.23 = 6.24×13<sup>−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"'> </span></span><![endif]><span style='font-size:14.0pt;font-family:Calibri'>Mole of HBTU needed=27 μmol; HBTU FW=379.3 g/mol; mass of HBTU needed=27×10<sup>−6</sup> mol×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"'> </span></span><![endif]><span style='font-size:14.0pt;font-family:Calibri'>Mole of DIPEA needed is 27 μmol. DPEA ρ=0.742 g/mL; FW=129.24 g/mol. Concentration of stock=0.742 g/mL÷129.24 g/mol=5.7×10<sup>−3</sup> mol/mL= 5.7 M. Volume of DIPEA needed= 27×10<sup>−6</sup> mol÷5.7 mol/L=4.7 μL.<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
−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]> <![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&WT.mc_id=keyname">SATA</a>-Aβ40
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 μmol Aβ40. <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
1–2 mL DMF (40 min) followed by DCM (1–2 mL, 40 min) and DMF
(1–2 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"'> </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"'> </span></span><![endif]><span style='font-size:14.0pt;font-family:Calibri'>Add the Kaiser reagents (2 drops or 15−20 μL)<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"'>
</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"'> </span></span><![endif]><span style='font-size:14.0pt;font-family:Calibri'>Heat at 100−120 °C for 4−6 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"'> </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 Aβ.<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"'>
</span></span><![endif]><span style='font-size:14.0pt;font-family:Calibri'>Mole of SATA needed=135 μmol; SATA FW=231.23 g/mol; mass of SATA needed=135×10<sup>−6</sup> mol×231.23 = 3.12×13<sup>−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"'>
</span></span><![endif]><span style='font-size:14.0pt;font-family:Calibri'>Mole of HBTU needed=135 μmol; HBTU FW=379.3 g/mol; mass of HBTU needed=135×10<sup>−6</sup> mol×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"'> </span></span><![endif]><span style='font-size:14.0pt;font-family:Calibri'>Mole of DIPEA needed is 135 μmol. DPEA ρ=0.742 g/mL; FW=129.24 g/mol. Concentration of stock=0.742 g/mL÷129.24 g/mol=5.7×10<sup>−3</sup>
mol/mL= 5.7 M. Volume of DIPEA needed= 135×10<sup>−6</sup>
mol÷5.7 mol/L=23.5 μL.<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
−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 μL TFA + 50 μL H<sub>2</sub>O + 50 μL
thioanisol + 25 μL EDT. For test-cleavage, use 30–60 μL
of this solution. Incubate shakingly for 3–4 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 μL of this
solution to 200 μL of 50% methanol in water and inject
150–170 μL 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>
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