After equilibration, place the Immobiline® DryStrip gels on filter paper moistened with deionized water. To help drain the equilibration solution, place the Immobiline® DryStrip gels so they rest on an edge. They can be left in this position for up to 10 min without noticeably affecting the spot sharpness. Alternatively, the Immobiline® DryStrip gels can be gently blotted with moistened filter paper to remove excess equilibration buffer.
Once the equilibrated Immobiline® DryStrip gels have drained for at least 3 min, use forceps to place them gel-side down on the ExcelGel through the slot at position 2 (Figure 54). The anodic side of the IPG DryStrip should be oriented such that it is toward the front edge of the gel.
Figure 53.Positioning the anodic buffer strip on Multiphor™ II unit.
Figure 54.Positioning equilibrated Immobiline® DryStrip gels on Multiphor™ II unit such that the anodic (acidic) side of the strip is toward the front edge of the gel.
Using forceps place one IEF sample application piece at the end of each Immobiline® DryStrip gel underneath the plastic “tab” formed by the overhanging gel support film at each end of the Immobiline® DryStrip gel. Be sure the application pieces touch the ends of the Immobiline® DryStrip gel (Figure 55).
Note: Application pieces absorb water that flows out of the Immobiline® DryStrip gels during electrophoresis.
Make sure that the Immobiline® DryStrip gel is in full, direct contact with the SDS gel. To remove any bubbles, stroke the plastic backing of the Immobiline® DryStrip gel gently with a spatula or forceps.
If loading marker proteins, place an extra application piece on the surface of the gel just beyond the end of the Immobiline® DryStrip gel. Pipette the markers onto the extra sample application piece. Apply the markers in a volume of 15–20 µL For less volume, cut the sample application piece proportionally. The markers should contain 200–1000 ng of each component for Coomassie staining and approximately 10–50 ng of each component for silver staining.
Place the IEF electrode holder on the electrophoresis unit, in the upper position, and align the electrodes with the center of the buffer strips. Plug in the electrode connectors and carefully lower the electrode holder onto the buffer strips (Figure 56).
Figure 55.Positioning application pieces.
Figure 56.Positioning electrodes.