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> <style> </style> </head>

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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>

Small-scale prepartion<o:p></o:p>

  1. 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>
  2. 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>
  3. Perform the Kaiser test<o:p></o:p>

<![if !supportLists]>a.    <![endif]>Take some resin out<o:p></o:p>

<![if !supportLists]>b.    <![endif]>Add the Kaiser reagents (2 drops or 15−20 μL)<o:p></o:p>

<![if !supportLists]>c.    <![endif]>Mix well by vortex.<o:p></o:p>

<![if !supportLists]>d.    <![endif]>Heat at 100−120 °C for 4−6 min<o:p></o:p>

<![if !supportLists]>e.    <![endif]>Assess the color change. Blue color suggests presence of free amines.<o:p></o:p>

  1. If free amines are present, there is no need for deprotection.<o:p></o:p>
  2. Calculate amount of SATA, HBTU and DIPEA. Need to use 10-fold excess to Aβ.<o:p></o:p>

<![if !supportLists]>a.    <![endif]>Mole of SATA needed=27 μmol; SATA FW=231.23 g/mol; mass of SATA needed=27×10−6 mol×231.23 = 6.24×13−3 g =6.2 mg <o:p></o:p>

<![if !supportLists]>b.    <![endif]>Mole of HBTU needed=27 μmol; HBTU FW=379.3 g/mol; mass of HBTU needed=27×10−6 mol×379.3 g/mol=10.24 mg<o:p></o:p>

<![if !supportLists]>c.    <![endif]>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=5.7×10−3 mol/mL= 5.7 M. Volume of DIPEA needed= 27×10−6 mol÷5.7 mol/L=4.7 μL.<o:p></o:p>

  1. Mix SATA, HBTU and DIPEA together and check the pH. <o:p></o:p>
  2. 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>
  3. Wash the resin profusely with DMF.<o:p></o:p>
  4. Wash the resin profusely with DCM.<o:p></o:p>
  5. Keep an aliquot for test cleavage.<o:p></o:p>
  6. Store the beads at −20°C or follow on to cleave the products using the CEM discover.<o:p></o:p>

<![if !supportEmptyParas]> <![endif]><o:p></o:p>

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>

Large-scale prepartion<o:p></o:p>

  1. 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>
  2. 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>
  3. Perform the Kaiser test<o:p></o:p>

<![if !supportLists]>a.    <![endif]>Take some resin out<o:p></o:p>

<![if !supportLists]>b.    <![endif]>Add the Kaiser reagents (2 drops or 15−20 μL)<o:p></o:p>

<![if !supportLists]>c.    <![endif]>Mix well by vortex.<o:p></o:p>

<![if !supportLists]>d.    <![endif]>Heat at 100−120 °C for 4−6 min<o:p></o:p>

<![if !supportLists]>e.    <![endif]>Assess the color change. Blue color suggests presence of free amines.<o:p></o:p>

  1. If free amino group is present there is not need for deprotection.<o:p></o:p>
  2. Calculate amount of SATA, HBTU and DIPEA. Need to use 5-fold excess to Aβ.<o:p></o:p>

<![if !supportLists]>a.    <![endif]>Mole of SATA needed=135 μmol; SATA FW=231.23 g/mol; mass of SATA needed=135×10−6 mol×231.23 = 3.12×13−2 g =31.2 mg<o:p></o:p>

<![if !supportLists]>b.    <![endif]>Mole of HBTU needed=135 μmol; HBTU FW=379.3 g/mol; mass of HBTU needed=135×10−6 mol×379.3 g/mol=51.2 mg<o:p></o:p>

<![if !supportLists]>c.    <![endif]>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=5.7×10−3 mol/mL= 5.7 M. Volume of DIPEA needed= 135×10−6 mol÷5.7 mol/L=23.5 μL.<o:p></o:p>

  1. Mix SATA, HBTU and DIPEA together and check the pH.<o:p></o:p>
  2. 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>
  3. Wash the resin profusely with DMF.<o:p></o:p>
  4. Wash the resin profusely with DCM.<o:p></o:p>
  5. Keep an aliquot for test cleavage.<o:p></o:p>
  6. Store the beads at −20°C or follow on to cleave the products using the CEM discover.<o:p></o:p>
  7. Cleave with cleavage solution 825 μL TFA + 50 μL H2O + 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>
  8. 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>

<a href="http://openwetware.org/wiki/Bitan:todo">Back to To-Do List</a><o:p></o:p>

<![if !supportEmptyParas]> <![endif]><o:p></o:p>

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