Biology Reference
In-Depth Information
2.3 Separation of
Enriched Membranes
by ZE-FFE
1. FFE system: BD™ FFE System (BD Diagnostics, NJ, USA,
for model years 2006-2010) or FFE System (FFE Service
GmbH, Germany, http://www.ffeservice.com/ , for model
years 2011-present).
2. Spacers and fi lters for ZE-FFE including 0.5 mm spacer and
0.8 mm electrode fi lter papers for ZE-FFE (FFE Service
GmbH, Germany).
3. 96-well deep-well plates (2 mL).
4. UV-transparent 96-well plates, e.g., UV-Star (Greiner Bio One,
NC, USA).
5. Microplate reader capable of reading absorbance at 280 nm,
e.g., Single-Mode Microplate Readers (Molecular Devices,
CA, USA).
6. FFE buffer 1: 280 mM sucrose, 10 mM acetic acid, 10 mM
triethanolamine, 1 mM EDTA, pH to 7.0 with NaOH
( see Note 4 ).
7. FFE buffer 2: 200 mM sucrose, 100 mM acetic acid, 100 mM
triethanolamine, 10 mM EDTA, pH to 6.5 with NaOH
( see Note 4 ).
8. FFE buffer 3: 100 mM acetic acid, 100 mM triethanolamine,
10 mM EDTA, pH to 6.5 with NaOH ( see Note 4 ).
9. Ultracentrifuge and fi xed-angle rotor with 10-15 mL tube
capacity, capable of 100,000 × g for sample concentration.
10. 10 mM Tris (hydroxymethyl) aminomethane (Tris-HCl), pH
7.5, stored at 4 °C.
1. High-grade trypsin, e.g., Trypsin, from Porcine pancreas
(Sigma-Aldrich, MO, USA).
2. SpeedVac concentrator.
3. Ultra-micro SpinColumns with C 18 (Harvard Apparatus,
MA, USA).
4. ACN1 solution: 80 % acetonitrile (v/v) with 0.1 % trifl uoro-
acetic acid (v/v).
5. ACN2 solution: 2 % acetonitrile (v/v) with 0.1 % trifl uoroacetic
acid (v/v).
2.4 Post-FFE
Sample Analysis
6. Tandem mass spectrometer (MS/MS) with online liquid
chromatography (LC) capabilities (nanofl ow or capillary fl ow
rates) capable of data-dependent acquisitions.
7. Search engine for analyzing mass spectrometry data to identify
proteins, e.g., Mascot (Matrix Science, UK).
8. Proteomic profi ling and quantitation software, e.g., Scaffold 3
(Proteome Software, OR, USA).
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