Protocol - Proteins extraction and trypsin digestion from subcutaneous abdominal fat
It is used in sample preparation for shotgun proteomics analysis
Category:
Biochemistry
Creation date: Feb 23, 2012
Last revision: Apr 16, 2015
Author(s):
Brambilla Francesca, Lavatelli Francesca, Di Silvestre Dario, Valentini Veronica, Rossi Rossana, Palladini Giovanni, Obici Laura, Verga Laura, Mauri Pierluigi, Merlini Giampaolo
Contact
Name: Pierluigi Mauri
Address: Institute for Biomedical Technologies (ITB-CNR), via Fratelli Cervi, 93, Segrate (MI)
Email: pierluigi.mauri@itb.cnr.it
Steps
| Description | Temperature | Time | Note |
|---|---|---|---|
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Obtain subcutaneous abdominal fat sample by fine needle aspiration.
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This procedure requires the acquisition of the informed consent from individuals.
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Weight the sample.
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For proteomics the weight of sample ranges between 10-20 mg.
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Wash the sample with sterile isotonic saline containing protease inhibitors.
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Crush with a disposable pestle in 100µl of lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT).
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Sonicate in ice-water with gentle shaking at room temperature between intervals.
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In ice/room temperature cicles
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Four times at intervals of 15 min
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Centrifuge at 25,000 x g
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4°C
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1h
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Remove the lipid content by recovering the central aqueous layer.
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Dialyze against 50 mM ammonium bicarbonate, using a 3.5 kDa cut-off membrane.
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4°C
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18h
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Digest the amount of protein determined by trypsin (1:50 enzyme:protein ratio).
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Incubate at 400 rpm
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37°C
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2h
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Terminate the digestion adding 1% v/v trifluoroacetic acid.
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Quality validation:
Yes
Validation info
The protocol has been used to produce data published in peer-reviewed journals.
Funded by: This research was supported by the Italian Ministry of Economy and Finance to the CNR for the Project FaReBio di Qualità and by the Fondazione Cariplo Nobel project, Proteomic platform, Operational Network for Biomedicine Excellence in Lombardy.
