Urinary Proteome Profiling Using 2D-DIGE and LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 293

(8) Formic acid ampoules, trifluoro-acetic acid ampoules
(Thermo Fisher Scientific, p/a Perbio Science, Northum-
berland)
(9) Plus One Repel Silane, Plus One Bind Silane, Dry Strip
Cover Fluid (Amersham Biosciences, GE Healthcare, NJ,
USA)
(10) 37.5:1 30% acrylamide-bis solution (Bio - Rad Laborato-
ries, CA)

2.1.Equipment

The 2D-DIGE equipment used in these experiments was pur-
chased from Amersham Biosciences, GE Healthcare, NJ, USA
(similar equipment may be purchased from other manufacturers):
(1) Immobiline Dry Strip Reswelling Tray
(2) Immobiline Dry Strip IEF gels
(3) Multiphor II Electrophoresis Unit
(4) Ettan DALT low-fluorescence glass plates with reference
markers
(5) Ettan DALT twelve gel caster, separation unit and power
supply
(6) Typhoon 9400 Imager
(7) Ettan Spot Picker
(8) DeCyder differential analysis software
(9) SpeedVac Thermo savant SPD 1010 (Thermo Fisher Sci-
entific, p/a Perbio Science, Northumberland)
(10) Agilent 1200 Series HPLC-Chip/MS system interfaced to
an Agilent 6210 TOF mass spectrometer (Agilent Tech-
nologies UK Limited, Life Sciences & Chemical Analysis
Group, Cheshire)

(1) Lysis buffer: 8 M urea, 2 M thiourea, 4% (w/v) CHAPS,
0.5% IGEPAL-630 (v/v), 10 mM Tris-HCl, pH to 8.3,
store at

2.2.Solutions

20 ◦ C.
(2) 10% stock detergent solution: 1 ml IGEPAL-630 (v/v)
Milli-Q 18

−

water volume adjusted to 10 ml, store at
room temperature.
(3) 40% stock (w/v) CHAPS solution: 40 mg CHAPS in
Milli-Q 18

water volume adjusted to 10 ml, store at
room temperature.
(4) CyDyes stock solutions: centrifuge at 12,000 × g for 30 s

prior to addition of 10 μ l DMF to 1000 pmol/ μ l, store

20 ◦ C. Before opening the tubes, stock solutions are
equilibrated on ice.
(5) L-lysine dye stop solution: 10 mM L-lysine in Milli-Q18

−

at

water, store at -20 ◦ C.