Team:Tuebingen/Notebook/Protocols/MALDI

From 2014.igem.org

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<h3>MALDI mass spectrometry analysis</h3>
<h3>MALDI mass spectrometry analysis</h3>
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<h4>Preparation of the samples for MALDI mass spectrometry</h4>
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<h4>Preparation of the samples for MALDI mass spectrometry analysis</h4>
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<ol>
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<ul>
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   <li>add 100 µl Buffer 2 (2 ml, 70 % 50 mM AB (ammoniumbicarbonate), 30 % ACN (acetonitrile)) to the Coomassie colored SDS gelpiece, incubate for 30 min, RT</li>
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   <li>add 100 µl Buffer 2 (2 ml, 70 % 50 mM AB (ammoniumbicarbonate), 30 % ACN (acetonitrile) to the Coomassie colored SDS gelpiece, incubate for 30 min, RT</li>
   <li>remove Buffer 2, add 100 µl ACN and incubate for 10 min, RT</li>
   <li>remove Buffer 2, add 100 µl ACN and incubate for 10 min, RT</li>
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   <li>remove ACN, add 100 µl DTT (10 mM dithiothreitol and 50 mM ammoniumhydrogencarbonate), incubate for 45 min, 56 °C</li>
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   <li>remove ACN, add 100 µl DTT (10 mM dithiothreitol and 50 mM ammoniumhydrogencarbonate),<br> incubate for 45 min, 56 °C</li>
   <li>remove DTT, add 100 µl IAA (55 mM iodacetamide and 50 mM AB), incubate for 30 min at RT with exclusion of light</li>
   <li>remove DTT, add 100 µl IAA (55 mM iodacetamide and 50 mM AB), incubate for 30 min at RT with exclusion of light</li>
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   <li>after 6 h add 2 µl 10 % TFA (trifluoracetate) to stop digestion</li>
   <li>after 6 h add 2 µl 10 % TFA (trifluoracetate) to stop digestion</li>
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</ol>
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</ul>
<p>&nbsp;</p>
<p>&nbsp;</p>
<h4>Measurement of the samples</h4>
<h4>Measurement of the samples</h4>
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<ol>
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<ul>
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   <li>add 1 µl matrix to goldplate, after that add 1 µl probe and mix them</li>
+
   <li>add 1 µl matrix to goldplate, after that add 1 µl of the sample and mix them</li>
   <li>after drying insert goldplate to MALDI massspec</li>
   <li>after drying insert goldplate to MALDI massspec</li>
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</ol>
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</ul>

Latest revision as of 01:35, 18 October 2014


Protocols

MALDI mass spectrometry analysis

Preparation of the samples for MALDI mass spectrometry analysis

  • add 100 µl Buffer 2 (2 ml, 70 % 50 mM AB (ammoniumbicarbonate), 30 % ACN (acetonitrile) to the Coomassie colored SDS gelpiece, incubate for 30 min, RT
  • remove Buffer 2, add 100 µl ACN and incubate for 10 min, RT
  • remove ACN, add 100 µl DTT (10 mM dithiothreitol and 50 mM ammoniumhydrogencarbonate),
    incubate for 45 min, 56 °C
  • remove DTT, add 100 µl IAA (55 mM iodacetamide and 50 mM AB), incubate for 30 min at RT with exclusion of light
  • remove IAA, add 100 µl Buffer 1 (50 mM AB), incubate for 15 min, RT
  • remove Buffer 1, add 100 µl Buffer 2, incubate for 10 min, RT
  • remove Buffer 2, add 100 µl ACN, incubate for 10 min, RT
  • remove ACN, add 20 µl Trypsin (β= 8.3 ng/µl)
  • after 6 h add 2 µl 10 % TFA (trifluoracetate) to stop digestion

 

Measurement of the samples

  • add 1 µl matrix to goldplate, after that add 1 µl of the sample and mix them
  • after drying insert goldplate to MALDI massspec

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