Pharmacia Biotech Multiphor II User Manual page 35

5. Operation
Apply the electrode strips to the long edges of the gel. Make sure the
electrode strips are applied with the correct polarity. Use sharp scissors to
cut off the strips which protrude beyond the ends of the gel.
Sample application
Some proteins may be pH sensitive (they may precipitate at specific pH
values) and better separation may be obtained when the sample is applied
at a specific position in the pH gradient. This can be tested by applying the
sample at various positions across the pH gradient.
There are three different methods for sample application. The method of
choice depends primarily by the sample and the volume to be applied.
1. IEF/SDS applicator strip for 5–20 µl sample volumes. This applicator
strip makes sample loading quick and simple, especially when a large
number of samples are to be applied. Check that the contact between the
gel and the applicator strip is uniform. Leave the applicator strip on the gel
during focusing.
2. Sample application pieces. Place the dry pieces on the Ampholine
PAGplate surface at the desired positions(s) in the gradient. Using a
micropipette, apply 15–20 µl volumes of sample solution on each piece. To
apply larger volumes, use 2 or 3 pieces, stacked or placed end to end, for
each sample applied. If smaller volumes are used, trim the paper
proportionally before applying it to the gel. Remove the pieces after
completing approximately half the total focusing time.
3. Very small sample volumes (e.g. 2 µl) can be applied as droplets directly
onto the dry gel surface.
To determine the pH gradient, prepare and apply pI markers according to
the instruction sheet included in the pI Marker Kit.
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