Application of DIGE and Mass Spectrometry in the Study of Type 2 Diabetes Mellitus Mouse Models - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 302

2.1.8. In-Gel Digestion and
NanoLC-MS/MS Analysis

1. Sample concentrator (SpeedVac; Thermo Fisher Scientifi c,
Hemel Hempstead, UK).
2. Dionex Ultimate 3000 with NCS-3500RS pump. NanoLC
system comprising vacuum degasser, nanocapillary pump,
loading pump, temperature-controlled column compartment,
autosampler and Chromeleon Express control software (Dionex,
Amsterdam, Holland).
3. LTQ-Orbitrap XL mass spectrometer with nanoelectrospray
(nESI) source and Xcalibur control software (Thermo Fisher
Scientifi c).
4. Acclaim PepMap 100 LC column, 75 mm ID × 15 cm, 3 m C 18
packing, and a precolumn cartridge (300 mm ID × 0.5 cm)
packed with Acclaim PepMap 100 (Dionex).
5. Proxeon ES508 stainless steel emitters (Thermo Fisher Scientifi c).
6. Proteome Discoverer 1.0 software (Thermo Fisher Scientifi c).
7. In-house Mascot search engine (Matrix Science, London, UK).

All solvents should be of HPLC-grade or equivalent unless stated
otherwise.

2.2. Consumables

1. Liquid nitrogen.
2. Lysis buffer at pH 8.3 (8 M urea, 2 M thiourea, 4% CHAPS
(w/w), 10 mM Tris, 0.5% NP40).
3. Protein Assay (Bio-Rad, Hemel Hempstead, UK).
4. Bovine serum albumin (BSA) standards in the range of 0-2 mg/
mL in water.
5. Disposable semi-microcuvettes.

2.2.1. Protein Extraction
from Mouse Liver Tissue

1. N , N -dimethylformamide, anhydrous (DMF) (see Note 1).
2. CyDye DIGE Fluor minimal labelling kit, 2 or 5 nmol (GE
Healthcare).
3. 10 mM solution of L -lysine monohydrochloride.
4. Lysis buffer at pH 8.3 (8 M urea, 2 M thiourea, 4% CHAPS
(w/w), 10 mM Tris, 0.5% NP40).

2.2.2. Protein Labelling

1. 1.3 M dithiothreitol (DTT; Sigma-Aldrich, Gillingham, UK).
2. IPG buffer with the same pH interval as the IPG strips
(3-10NL; GE Healthcare).
3. DeStreak rehydration solution (GE Healthcare).
4. DryStrip cover fl uid (GE Healthcare).
5. IPGphor strip holder cleaning solution (GE Healthcare).

2.2.3. Rehydration
of IPG Strips

1. Lint-free wipes.
2. DryStrip cover fl uid (GE Healthcare).
3. IPGphor strip holder cleaning solution (GE Healthcare).

2.2.4. Isoelectric Focusing