Application of DIGE and Mass Spectrometry in the Study of Type 2 Diabetes Mellitus Mouse Models - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 303

2.2.5. Second-Dimension
Electrophoresis

1. SDS-PAGE gels.
2. DTT.
3. Iodoacetamide (IA; GE Healthcare).
4. Equilibration buffer (aqueous solution containing 50 mM
Tris, 6 M urea, 30% glycerol, 2% sodium dodecyl sulphate).
5. Agarose (ReadyPrep Overlay Agarose; Bio-Rad).
6. Tris-glycine-SDS run buffer, 10 times concentrate (National
Diagnostics Protogel, obtainable from Fisher Scientifi c,
Loughborough, UK).

1. Water for plate cleaning. 2.2.6. Gel Scanning

and Image Analysis

1. Fixing solution (40% ethanol, 10% acetic acid).
2. Destaining solution (1% acetic acid).
3. Ammonium sulphate.
4. Brilliant Blue G250.
5. Phosphoric acid.
6. Methanol.
7. Ethanol.

2.2.7. Visualisation
and Picking of Proteins
of Interest

3. Methods

To exclude the possibility of sample contamination with non-mouse
proteins or other contaminants, it is advisable to carry out as
many of the laboratory procedures as possible in a designated clean
room or area. If this is not possible, great care must be taken at
every step to avoid contamination, especially from human keratin
which tends to be ubiquitous.

3.1. Protein Extraction
from Mouse Liver
Tissue

This protocol assumes that mouse liver tissues are frozen at −80°C.
It is critically important to keep the liver tissues frozen until the
lysis buffer has been added to the ground samples, to prevent pro-
teases degrading the proteins.
The fact that liver tissue samples contain high levels of protein
gives the advantage that it is not necessary to undertake additional
sample clean-up steps. By taking only a small quantity of the liver lysate
for DIGE, levels of possible interfering compounds will be low:

1. Add the mouse liver tissue to the pre-cooled mortar containing
liquid nitrogen. Grind the frozen tissue to a fi ne powder, topping
up the liquid nitrogen as it evaporates so that the tissue does
not thaw.