Evaluating the Efficacy of Subcellular Fractionation of Blast Cells Using Live Cell Labeling and 2D DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 328

lay the strip down such that the gel is facing the solution.
Ensure the buffer is evenly distributed under the strip. Add
1 mL of cover fl uid on top of the strip to prevent dehydration
of strip and evaporation of the buffer. Incubate strips over-
night at room temperature.

2. After CyDye labeling of the samples, add 0.75 mL of IPG buffer

pH 3-11 NL, 1.5 mL of 1 M DTT stock solution, and 0.75 mL
of BPB solution, then make up the volume of each sample to
150 mL with lysis buffer.

3. Position the ceramic IPG strip tray on the Ettan IPGphor II

(GE Healthcare). Align a rehydrated IPG strip along the groove
inside the manifold with the gel side facing up. Soak two paper
wicks per IPG strip in 150 mL of deionized water. Place one wet
paper wick between the IPG strip and the anode and the other
between the cathode and the IPG strip. Position the loading
cup right below the anode wick and click in using the IPG cup
pusher to form a good seal between the cup and the strip. Cover
the IPG strips and around the cups by pouring 100 mL of cover
fl uid into the manifold. Ensure the cups are not leaking. Add
the 150 mL of sample into the cup. Cover the sample with
50 mL of cover fl uid to prevent sample from drying out.

4. Conduct isoelectric focusing according to the protocol outlined

in Table 1 .

5. Prepare the DTT and IAA equilibration solutions. Place the

IPG strip in a long, rod-shaped, plastic tube. Incubate the IPG
strip in the DTT equilibration solution for 15 min on an orbital
shaker in a fl at position. During this incubation time, prepare
the anode and cathode buffers.

6. Pour off the DTT equilibration solution and add the IAA

equilibration solution and incubate for 15 min at room tem-
perature with gentle agitation. Cover solution with aluminum

Table 1
Isoelectric focusing program on the IPGPhor II

Step Type Voltage (V) Duration (h) Accum (Vh)

1 Step 300 2 600

2 Step 500 2 1,600

3 Step 1,000 2 3,600

4 Gradient 8,000 5 28,600

5 Step 8,000 5 68,600

6 Step 500 10 -