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Team:Tuebingen/Notebook/Protocols/MALDI
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<ul> | <ul> | ||
| - | <li>add 100 µl Buffer 2 (2 ml, 70 % 50 mM AB (ammoniumbicarbonate), 30 % ACN (acetonitrile | + | <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> | ||
| - | <li>remove ACN, add 100 µl DTT (10 mM dithiothreitol and 50 mM ammoniumhydrogencarbonate), incubate for 45 min, | + | <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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<h4>Measurement of the samples</h4> | <h4>Measurement of the samples</h4> | ||
<ul> | <ul> | ||
| - | <li>add 1 µl matrix to goldplate, after that add 1 µl | + | <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> | ||
</ul> | </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
