Bitan:Microwave-assistant peptide synthesis

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<p class=MsoNormal style='tab-stops:87.2pt'><b><span style="mso-spacerun: yes">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span><span style="mso-spacerun: yes">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span style="mso-spacerun: yes">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span style="mso-spacerun: yes">&nbsp;&nbsp;</span><a href="http://openwetware.org/wiki/Bitan:todo">[Back to Bitan: todo page]</a><o:p></o:p></b></p>

<p class=MsoNormal style='tab-stops:87.2pt'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></p>

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 <td width=935 valign=top style='width:935.1pt;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal align=center style='text-align:center'><span
 style='font-size:16.0pt;color:#993300'><b>Microwave-assistant peptide
 synthesis</b></span><span style='font-size:16.0pt;color:#993300;mso-fareast-language:
 ZH-CN'> <o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Instrument Type:
 Discover SPS - Manual Peptide Synthesis </span><b><o:p></o:p></b></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>(The method is
 modified from the CEM discover SPS installation instruction)<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><b>1. Install the
 instruments as manual book.<o:p></o:p></b></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><b><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></b></span></p>
 <p class=MsoNormal style='tab-stops:432.8pt'><span style='mso-fareast-language:
 ZH-CN'><b>2. Create a microwave method.<span style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></b></span><span
 style='mso-fareast-language:ZH-CN'><o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>1) Press the Open
 Folder Button.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>2) Select “New
 method”.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>3) Press the
 right arrow key until “ Mode = Discover SPS”. Then press ENTER.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>4) Set power then
 press ENTER.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>5) Set maximum temperature
 then press ENTER.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>6) Set Run time,
 then press ENTER.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>7) Set delta
 temperature then press ENTER. (Delta temperature is the minimum value the
 temperature must drop below the maximum temperature before the power will be
 re-applied. Defult = 5°C).<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>8) Set stirring
 to OFF, then press ENTER.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>9) Set cooling to
 OFF press ENTER.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>10) Set “next
 stage = (N)” then press ENTER.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>11) Set “save
 method = (Y)” then press ENTER.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>12) Create method
 name using the arrow keys and highlight “ Exit” and press ENTER when done.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>13) The method is
 now saved in the software. To load different methods, press Open Folder
 Button and use the arrow keys to scroll through available methods.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><b>3. Recommended
 parameters for Fmoc Solid Phase Synthesis<o:p></o:p></b></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>1) Fmoc
 deprotection<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Power = 20 W; Temperature
 = 75 °C; Time = 3 min; Delta Temp. = 5°C<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>2) Coupling <o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Power = 20 W;
 Temperature = 75 °C; Time = 5 min; Delta Temp. = 5°C<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>3) TFA cleavage<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Power = 20 W;
 Temperature = 38 °C; Time = 18 min; Delta Temp. = 5°C<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><b>4. Prepare reaction
 vessel, dry solvents (DMF, DCM, and MeOH), resin, and activation reagents et
 al.<o:p></o:p></b></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><b>5. Synthesis
 steps<o:p></o:p></b></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>1) Place the luer
 plug on the bottom of reaction vessel and swell the resin in reaction vessel
 in DMF for around 1 hour before experiment. <o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>2) Turn on the
 vacuum pump. Place the reaction vessel in front of the waste bottle
 immediately after removing the luer plug. The liquid waste should drain into
 the 1L bottle. <o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>3) Wash the resin
 with DMF (5&times;). Allow the liquid to drain and turn off the vacuum pump.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>4) Remove the
 reaction vessel from the vacuum manifold and reattach the lure plug. The
 resin is ready to use now. <o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>5) If the resin
 is pre-protected by Fmoc, remove Fmoc with deprotection solvent (deprotection
 solvent:<span style="mso-spacerun: yes">&nbsp; </span>20% piperidine + DMF).
 Add the deprotection solvent to resin. Shake for 20 min in room temperature. <o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>6) Turn on the
 vacuum pump. Place the reaction vessel in front of the waste bottle
 immediately after removing the luer plug. Wash the resin with DMF (5&times;).
 Allow the liquid to drain and turn off the vacuum pump.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>7) First amino
 acid coupling: <o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>i) Prepare the
 coupling amino acid activation solution. (Recommend amino acid/HBTU/DIEA
 method for normal peptide synthesis). Equimolar amounts of protected amino
 acid and HBTU are dissolved in DMF (usually a 3-fold excess). 6-fold excess
 of base (DIPEA) is added after the solution is stirred for 10 minutes, and
 then added to the reaction vessel. <o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>ii) Insert the
 fiber-optic probe into the thermowell. Insert the thermowell into the clip,
 and attach the clip to the reaction vessel.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>iii) Place the
 reaction vessel into the holder and insert into the microwave cavity.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>iv) Press the
 play key on the panel and the loaded coupling method will run.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>8) After the
 method running is done, cool down the solution to the set temperature. <o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>9) Wash the resin
 with DMF (5&times;). Allow the liquid to drain and turn off the vacuum pump.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>10) Test the
 coupling efficiency. (For difficult sequence, the method of test cleavage
 detected by Mass spectrometer or Fmoc UV detection (see procedure 1 below) is
 recommended. For normal sequence TNBS test or Kaiser test is recommended (see
 procedure 2 and 3 below). <o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>11) If the
 coupling is efficient, deprotect the Fmoc group as step 5.5. If the coupling
 is not complete, repeat the coupling step with fresh prepared the amino acid
 activation solution.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>12) Repeat
 coupling and deprotection Fmoc steps to the end of the sequence. <o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><b>6. Cleavage<o:p></o:p></b></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>1) Prepare the
 resin: Wash the resin completely with DMF and then DCM. Dry the resin in
 vacuum until it is completely dry. <o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>2) Prepare the
 cleavage cocktail. Different sequence or resin need different cleavage
 cocktail. Reagents R (TFA/phenol/water/thioanisole/EDT (82.5/5/5/5/2.5)) and
 B (TFA/phenol/water/TIPS (88/5/5/2)) are compatible with most sequences, and
 are strongly recommended<i> </i></span><span style='mso-fareast-language:
 ZH-CN'>for sequences containing Trp, His, Met, Cys, Arg, Gln, or Asn, as well
 as for peptides constructed on a PAL or Rink Amide resin. For other special
 resin, please look up the resin instruction from the company.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>3) Add the
 cleavage solution to resin. Be sure you wear protection (gloves and goggles)
 when you handle TFA. <o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>4) Since our
 microwave system installed with open vessel, we cannot do cleavage under
 microwave in our system. They have new cleavage system available in CEM
 Company, but we would like to do this step in room temperature. After add the
 cleavage solution to the dry resin, seal the vessel and shake for less than 2
 hours. <o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>5) Collect the
 product after cleavage:<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>i) Insert a 15 mL
 centrifuge tube inside the 250 mL bottle and lid is secure.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>ii) Turn on the
 vacuum pump.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>iii) Place the
 reaction vessel in the well located in front of the 250 mL bottle immediately
 after removing the luer plug. <o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>iv) Wash the
 resin with a small amount of cleavage solution or pure TFA to ensure all
 cleaved peptide has been transferred to the centrifuge tube.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>v) Turn off the
 vacuum pump.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>vi) Remove the
 reaction vessel and discard.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>6) Take the 15 mL
 tube with the collected peptide solution out of the bottle.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><b>7.
 Precipitation<o:p></o:p></b></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>1) Concentrate
 the collected peptide solution with a strain of high pure N<sub>2</sub> gas
 to 1&#8211;2 mL.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>2) Add cold ether
 to the concentrated solution and sit the tube in ice or freezer. The crude
 peptide will precipitate. <o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>3) Wait couple of
 hours or just leave the tube in freezer overnight. Centrifuge the solution
 and collect the precipitate. <o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>4) Add cold ether
 and centrifuge again to remove the soluble impurities. Repeat 2&#8211;3
 times. <o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>5) Collect the
 precipitate and dry it under vacuum.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>6) Now you have your
 crude peptide! <o:p></o:p></span></p>
 <p class=MsoNormal><![if !supportEmptyParas]>&nbsp;<![endif]><span
 style='mso-fareast-language:ZH-CN'><o:p></o:p></span></p>
 </td>
</tr>

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<p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></span></p>

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 <td width=611 valign=top style='width:611.1pt;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><b>Procedure 1:
 Fmoc-quantitation<o:p></o:p></b></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>The cuvette 1 is
 filled with 3 ml of the sample solution (Fmoc deprotection solution with a
 certain dilution). <o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>The cuvette 2 is filled
 with 3 ml of the blank solution (blank solution is prepared in the same
 manner but without addition of the resin). <o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>The cuvette 3 is
 filled with 3 ml of the reference solution (reference solution is prepared in
 the same manner but with resin known loading rate)<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>A UV spectrometer
 is set to zero at 290 nm on the blank solution, and the optical density of
 the sample and reference solution measured. <o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>The cuvettes are
 emptied and cleaned and the measurement repeated twice with fresh solutions.
 The loading is calculated by compare the UV absorption of sample solution and
 reference solution.<o:p></o:p></span></p>
 <p class=MsoNormal><![if !supportEmptyParas]>&nbsp;<![endif]><span
 style='mso-fareast-language:ZH-CN'><o:p></o:p></span></p>
 </td>
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<p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></span></p>

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 <td width=611 valign=top style='width:611.1pt;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><b>Procedure 2:
 TNBS test<o:p></o:p></b></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Solution 1: 5%
 DIPEA in DMF<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Solution 2: 1%
 aqueous TNBS<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>A few resin beads
 are placed in a small test tube and 1-3 drops of each solution are added. <o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>The test is
 positive when the resin beads turn yellow or red within 10 min and negative
 when the beads remain colorless.<o:p></o:p></span></p>
 <p class=MsoNormal><![if !supportEmptyParas]>&nbsp;<![endif]><span
 style='mso-fareast-language:ZH-CN'><o:p></o:p></span></p>
 </td>
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<p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></span></p>

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 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><b>Procedure 3:
 Kaiser Test<o:p></o:p></b></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Solution 1: 5 g ninhydrin
 in 100 mL ethanol<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Solution 2: 80 g
 phenol in 20 ml ethanol<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Solution 3: 2 mL
 0.001 M aqueous KCN in 98 mL pyridine<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>A few resin beads
 are placed in a small test tube and 2-5 drops of each solution are added.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>The tube is
 placed in an oven and the reaction left to develop for 5 min at 100°C.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>The test is
 positive when the resin and solution turn blue and negative when the beads
 remain colorless.<o:p></o:p></span></p>
 <p class=MsoNormal><![if !supportEmptyParas]>&nbsp;<![endif]><span
 style='mso-fareast-language:ZH-CN'><o:p></o:p></span></p>
 </td>
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</table>

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<p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><b>Recommended reading:<o:p></o:p></b></span></p>

<p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></span></p>

<p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>1. The microwave-assistant peptide synthesizer used in our lab:<span style="mso-spacerun: yes">&nbsp; </span><a href="http://www.cem.com/page11.html">http://www.cem.com/page11.html</a><o:p></o:p></span></p>

<p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>2. Stacey A. Palasek, Zachary J. Cox, Jonathan M. Collins. <i>J. Pept. Sci. </i></span><span style='mso-fareast-language:ZH-CN'>2007; <b>13</b></span><span style='mso-fareast-language:ZH-CN'>: 143&#8211;148<o:p></o:p></span></p>

<p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>3. Introduction of Fmoc solid phase peptide synthesis method and procedures </span><a href="http://www.chempep.com/ChemPep-Fmoc-Solid-Phase-Peptide-Synthesis.htm">http://www.chempep.com/ChemPep-Fmoc-Solid-Phase-Peptide-Synthesis.htm</a></p>

<p class=MsoNormal><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></p>

<p class=MsoNormal><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></p>

<p class=MsoNormal><b><a href="http://openwetware.org/wiki/Bitan:todo">[Back to Bitan: todo page]</a><o:p></o:p></b></p>

<p class=MsoNormal><b><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></b></p>

<p class=MsoNormal><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></p>

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