Evaluating the Efficacy of Subcellular Fractionation of Blast Cells Using Live Cell Labeling and 2D DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 323

13. Lysis buffer: 7 M urea, 2 M thiourea, 4% (w/v) 3-[(3-cholami-
dopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),
and 30 mM Tris in water at pH 9.5. Store aliquots at −20°C
and thaw on ice when required. Once this lysis buffer is mixed
with the protein sample, the pH will decrease to at about pH
8.5.
14. EZQ protein quantitation kit (Invitrogen).
15. EZQ protein quantitation reagent (Component A). Store pro-
tected from light.
16. EZQ rinse buffer: 10% methanol and 7% acetic acid in water.
17. Plastic staining tray.
18. pH indicator paper, pH 8.2-10.
19. Ovalbumin protein quantifi cation standard: Prepare a 2 mg/
mL stock solution by solubilizing 2 mg provided with EZQ
protein quantitation kit in 1 mL of lysis buffer (see Subheading
2.3 , item 10). Store 10-mL aliquots at −80°C and thaw on ice
when required.
20. Typhoon Trio fl uorescent scanner (GE Healthcare).

2.4. Two-Dimensional
Electrophoresis

1. Rehydration buffer: 7 M urea, 2 M thiourea, 30 mM Tris, 2%

(w/v) CHAPS, 0.5% (v/v) IPG buffer pH 3-11 NL (GE
Healthcare), 1.2% (v/v) DeStreak reagent (GE Healthcare),
and 0.05% (v/v) saturated Bromophenol blue solution.

2. 24-cm IPG strips, pH 4-7 (GE Healthcare). Store at −20°C.

3. PlusOne dry strip cover fl uid (GE Healthcare). Store at room

temperature.

4. Strip holder cleaning reagent (GE Healthcare). Store at room

temperature.

5. Reswelling tray (GE Healthcare).

6. Paperwicks (GE Healthcare).

7. Loading Cups (GE Healthcare).

8. Ceramics IPG strip tray (GE Healthcare).

9. IPGPhor II isoelectric focusing apparatus (GE Healthcare).

10. 2DGel DALT 12.5% NF (Serva, Heidelberg, Germany). Store
at 4°C.

11. DTT solution: To make 1 M solution, weigh 0.1542 g of DTT

(Sigma-Aldrich) and dissolve in 1 mL of water. Mix well. Store
aliquots at −80°C and thaw at room temperature when required.
12. DTT equilibration solution: Dissolve 0.05 g of DTT and 1.8 g
urea in 5 mL of IPG equilibration buffer (Serva). This recipe is
designed for one strip. Adjust the amount and volume accord-
ing to the number of strips.