Biology Reference
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3.
Soak two paper wicks per IPG strip in 150 mL of reverse osmosis
water (see Note 7). Place the wet paper wick over each end of
the IPG strip such that the wick covers approximately one-
third of the end of the gel, with two-thirds extending over the
plastic backing.
4.
Position the loading cup immediately below the paper wick at
the anodic end of the strip. Pour 100 mL of cover fl uid into
the manifold ensuring the strips are covered but do not pour
into the cups. Observe that the cups are correctly sealed against
the IPG strips and do not fi ll with cover fl uid. If the cups are
not leaking, gently pipette the samples into the cups taking
care not to introduce any bubbles. Cover the samples with
50 mL of cover fl uid to prevent drying during the run.
5.
Place the electrodes on the paper wicks such that two-thirds of
the wick is positioned in front of the electrode wire and one-
third behind.
6.
Perform IEF according to the program outlined in Table
1
.
7.
Prepare the DTT and IAA equilibration solutions. Place a
maximum of two focused IPG strips into a single, long, rod-
shaped, plastic tube (e.g., the tubes that sample cups are pro-
vided in from GE Healthcare). Incubate the IPG strips in the
DTT equilibration solution for 15 min in a fl at position with
gentle agitation. During this incubation time, prepare the
anode and cathode buffers.
8.
Decant the DTT equilibration solution and replace with IAA
equilibration solution. Cover the tubes with aluminum foil and
incubate for 15 min at room temperature with gentle agita-
tion. Remove the IAA equilibration solution and add 5 mL of
cathode buffer per tube to rinse off the equilibration solution.
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 -
The purpose of step 6 is to maintain the electric fi eld overnight and prevent
protein diffusion
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