Biology Reference
In-Depth Information
1. Rehydration buffer: 8 M Urea, 2 % (w/v) CHAPS, 0.05 %
(w/v) bromophenol blue. Just prior to use add 0.5 % (v/v)
IPG buffer (pH 3-10 NL, GE Healthcare Life Sciences) and
18 mM DTT (
see
Note 10
).
2. Vortex and microcentrifuge such as Eppendorf 5430 or an
equivalent.
3. Ettan ceramic strip holder or equivalent (GE Healthcare Life
Sciences) for IEF equipment being used.
4. Immobiline DryStrips 3-10 NL 240 mm (GE Healthcare Life
Sciences) or equivalent.
5. IPG cover fl uid (GE Healthcare Life Sciences) or mineral oil
equivalent.
6. Ettan IPGphor 3 (GE Healthcare Life Sciences) or an equivalent.
Isoelectric Focusing
1. 12 % SDS-PAGE gel solution: 375 mM Tris-HCl, pH 8.8,
0.1 % (w/v) SDS, 0.36 % (w/v) bisacrylamide, 12 % (w/v)
acrylamide, with 0.1 % (w/v) AMPS, 0.04 % (v/v) TEMED
being added just before use.
2. Forceps.
3. Equilibration buffer: 50 mM Tris-HCl, pH 8.8, 6 M urea, 2 %
(w/v) SDS, 26 % (v/v) glycerol, 0.05 % (w/v) bromophenol blue.
DTT (65 mM) or IAA (135 mM) are added freshly before use.
Transferring and Setting
IEF Strips onto SDS-PAGE
4. Rinse solution: 1.5 M Tris-HCl pH 8.8, 1 % (w/v) SDS.
5. IPG fi xing solution: 375 mM Tris-HCl, pH 8.8, 1 % (w/v)
SDS, 0.5 % (w/v) agarose, 0.5 % (w/v) bromophenol blue.
1. Software analysis packages such as DeCyder 2D Software
(GE Healthcare Life Sciences) or DeCodon Software
(DECODON).
Gel Scanning and Software
Analysis
1. V-bottom 96-well plate made of polypropylene such as Greiner
Bio One (651201) and sealing fi lm such as Nunc (232702) or
equivalent.
2. Destain solution: 50 % (v/v) ACN, 10 mM NH
4
HCO
3
.
3. Orbital shaker.
4. Dry block heater.
5. Digestion solution: 12
Extraction and Digestion
of Proteins That Differ
in Abundance on DIGE
g/mL Trypsin, 10 mM NH
4
HCO
3
,
0.0012 % TFA (
see
Notes 11
and
12
).
6. 37 °C heating oven.
μ
A. Method using MALDI-MS/MS.
1. Peptides extracted from a protein spot as outlined in
Subheading
3.4.4
.
2. MALDI mass spectrometry calibration standards.
Identifi cation of Proteins That
Differ in Abundance on DIGE
