Evaluating the Efficacy of Subcellular Fractionation of Blast Cells Using Live Cell Labeling and 2D DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 326

bonds. Separate cell lysate using a NuPAGE 4-12% Bis-Tris gel
in MES SDS running buffer at 180 V and 100 mA/gel until
the dye front reaches the bottom of the gel (~45 min). Image
gel using the Typhoon Trio (GE Healthcare) with laser excita-
tion at 532 nm and emission fi lter at 580 nm. Detectable bands
indicate the labeling process has been successful.

1. Transfer cells into a 2-mL microcentrifuge tube. Spin cells at
400 × g for 2 min at room temperature. Remove supernatant
carefully and completely without disturbing the pellet.
2. Add 10 mL of protease inhibitor cocktail to the wall of the tube
and immediately add 2 mL of ice-cold extraction buffer I.
Resuspend the cell pellet carefully and thoroughly by pipetting.
Incubate tube for 10 min at 4°C on a rotary shaker.
3. Spin tube at 16,000 × g at 4°C for 15 min to pellet insoluble
material. Transfer supernatant to a new 15-mL Falcon tube
labeled cytosolic proteins. Keep fraction on ice for same day
use. Protein can be stored at −20°C at this stage.
4. Add 5 mL of protease inhibitor cocktail to the wall of the tube
and immediately add 1 mL of ice-cold extraction buffer II to
the cell pellet. Mix thoroughly by pipetting. Incubate tube at
4°C for 30 min on a rotary shaker.
5. Spin tube at 16,000 × g at 4°C for 15 min. Transfer supernatant
to a new 15-mL Falcon tube labeled membrane proteins. Keep
fraction on ice for same day usage. Otherwise, store at −20°C
(see Note 2).
6. To concentrate the proteins used in 2D DIGE, the ProteoExtract
protein precipitation kit is used. Mix by briefl y vortexing the
sample with four volumes of ice-cold precipitation agent.
Incubate tube overnight at −20°C. Spin down the precipitated
proteins at room temperature for 10 min at 10,000 × g .
7. Carefully remove supernatant completely without disturbing
the pellet. Wash the protein pellet by resuspending in 1 mL of
fresh ice-cold wash solution and vortex briefl y. Spin tube for
2 min at room temperature at 10,000 × g .
8. Carefully remove supernatant completely without disturbing
the pellet. Repeat the wash step in 0.5 mL of ice-cold wash
solution. Aspirate the supernatant without disturbing the pellet.
Air-dry the pellet for 1 h at room temperature.
9. Resuspend protein lysate in a small volume of lysis buffer to
start with (i.e., 10 mL). Add the volume of lysis buffer in a
small interval until no visible precipitated protein is observed.
Sonicating in ice-cold water bath greatly improves resolubiliza-
tion of protein lysate.

3.3. Proteomic
Extraction and
Labeling of Membrane
and Cytoplasmic
Fractions