2D DIGE for the Analysis of RAMOS Cells Subproteomes - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 243

2.8. Scanning Gels
and Analyzing

1. Typhoon 9400 (GE Healthcare).

2. DeCyder V7.0 Software (GE Healthcare).

3. Methods

Both cell conditions were grown in parallel, and protein extracts
from the nuclear, membranes, and cytosolic fractions were pre-
pared as described in the protocol for suspension culture cells,
included in the ProteoExtract™ Subcellular Proteome Extraction
Kit (Calbiochem, San Diego, USA) but with a greater number of
cells for the subsequent analysis.
Briefl y, cells in their logarithmic growth phase were centri-
fuged and washed twice with 2 mL of ice-cold wash buffer, at
300 × g and 4°C for 10 min. The cell pellet was resuspended in
1 mL of ice-cold extraction buffer I and 5 mL of protease inhibitor
cocktail, incubated for 10 min at 4°C under gentle agitation and
centrifuged at 1,000 × g and 4°C for 10 min. The supernatant was
Fraction F1, and the cell pellet was resuspended in 1 mL of ice-
cold extraction buffer II and 5 mL of protease inhibitor cocktail,
incubated for 30 min at 4°C under gentle agitation and centri-
fuged at 6,000 × g and 4°C for 10 min. The supernatant was
Fraction F2, and the cell pellet was resuspended in 500 mL of
extraction buffer III with 5 mL of protease inhibitor cocktail and
1.5 mL (>375 U) of benzonase, incubated for 10 min at 4°C
under gentle agitation and centrifuged at 6,800 × g and 4°C for
10 min. The supernatant was Fraction F3, and the cell pellet was
resuspended in 500 mL of room temperature extraction buffer IV
and 5 mL of protease inhibitor cocktail to give Fraction F4 (see
Note 1).

3.1. Subcellular
Fractionation

3.2. Sample
Preparation

One of the most important steps is the sample preparation which
includes the elimination of all the contaminants present that
could interfere with the electrophoresis, such as salts, detergents,
nucleic acids, lipids, polysaccharides, phenols, etc. In this experi-
ment in particular, the reagents of the kit used in Subheading 3.1
are incompatible with electrophoresis. Due to this fact, the
following chloroform-/methanol-based protein precipitation
protocol was used:

1. 1 mL of Fractions F2 and F3 are divided into 250 mL aliquots.

It is recommended to perform the entire process in 1.5- or
2.0-mL polypropylene tubes.

2. Add 3 volumes of methanol and 1 volume of chloroform and

vortex.

3. Add 3 volumes of Milli-Q water and vortex well.