Evaluating the Efficacy of Subcellular Fractionation of Blast Cells Using Live Cell Labeling and 2D DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 322

2.2. Live Cell Labeling

1. PBS buffer (see Subheading 2.1 , item 3).

2. Binding buffer: 7 mM dithiothreitol (DTT) and 1 M urea in

Hank's Balanced Salt Solution (HBSS) (Invitrogen), pH 8.5.
Keep solution at 4°C.

3. CyDye DIGE fl uor Cy3 minimal dyes (GE Healthcare,

Uppsala, Sweden): 600 pmol CyDye working solution per
labeling in anhydrous 99.8% N,N -Dimethylformamide (DMF).
Store protected from light at 4°C for current use, otherwise
store aliquots at −80°C (see Note 3).

4. 10 mM L -lysine. Store at −20°C.

5. NuPAGE 4-12% Bis-Tris gel (Invitrogen).

6. 2× LDS buffer: Dilute NuPAGE 4× LDS sample buffer

(Invitrogen) 1:1 with water.

7. MES SDS running buffer: 50 mM MES pH 7.2, 50 mM Tris-

base, 0.1% SDS, and 1 mM EDTA in water (pH 7.3; do not
adjust with acid or base).

2.3. Protein
Preparation
(Fractionation)
and Labeling

1. ProteoExtract native membrane protein extraction kit (Calbio

chem, Merck KGaA, Darmstadt, Germany). Store kit at 4°C.

2. Extraction buffer I (provided in extraction kit (see Subheading

2.3 , item 1)): Thaw and mix well before use.

3. Extraction buffer II (provided in extraction kit (see Subheading

2.3 , item 1)): Thaw and mix well before use.

4. Protease inhibitor cocktail (provided in extraction kit (see

Subheading 2.3 , item 1)): Thaw and mix well before use. Keep
at room temperature until needed.

5. ProteoExtract protein precipitation kit (Calbiochem).

6. Precipitation agent: To one bottle of Precipitant 1, add 1.7 mL

of Precipitant 2, 1.7 mL of Precipitant 3, and 1.7 mL of
Precipitant 4. Mix well. Store precipitation agent at −20°C for
up to 2 months.

7. Wash solution: Add 150 mL of ethanol to the provided wash

solution (Calbiochem ProteoExtract protein precipitation kit).
Mix well. Store at −20°C up to 1 year.

8. CyDye DIGE fl uor Cy5 minimal dye (GE Healthcare):

200 pmol CyDye working solution per labeling in DMF. Store
protected from light at 4°C for current use, otherwise store
aliquots at −80°C (see Note 3).

9. 10 mM L -lysine (see Subheading 2.2 , item 4).

10. 1.5-mL Microcentrifuge tubes.
11. 2-mL Microcentrifuge tubes.
12. 15-mL Falcon polypropylene conical centrifuge tube (BD)
(see Note 2).