Application of 2D DIGE in Animal Proteomics - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 391

17. We have made good experiences with the stability of the DMF-
dissolved CyDyes, even beyond the recommended shelf-life,
provided that the dyes are always properly stored and only
removed from the freezer for short intervals. Take special care
to always use fresh pipette tips in order to avoid contamina-
tions. When using larger packages, aliquot the dissolved dyes
and use them one by one.
18. For larger sample volumes, the volumes of the stop solution and
the IPG sample buffer are increased accordingly. However, make
sure that overall sample volumes are not getting too large (after
mixing up to three single samples), as this protocol describes point
application via sample application pieces on the IPG strips.
19. The ratio for dye-to-protein labeling recommended by the
manufacturer is 200 pmol dye/25 mg protein. However, this is
minimal labeling, and works also with lower amounts of dyes.
If DIGE serves only for analytical purposes, both dye and pro-
tein amounts may be cut down, as seen for instance in Fig. 3 .
20. The supporting fi lm should stick fi rmly to the back of the
casting cassette. Too much water, large air bubbles, or small
particles trapped below the fi lm make it camber, thus affecting
gel thickness.
21. If smaller gels (fewer IPG strips) are needed, a casting cassette
of the same dimensions but with a U-frame open at the shorter
side (“portrait format”) is commercially available (e.g., from
GE Healthcare).
22. Gradients can also be poured without a peristaltic pump, just
by gravity. Liquid fl ow is then controlled by differences in
height (putting gradient maker and magnetic stirrer well above
the casting cassette), as shown in Fig. 4 of ( 17 ). The once
established setup has to be used consistently to ensure repro-
ducibility in casting.
23. Avoid getting bubbles into your gel cassettes, as they will form
holes in the gel. However, if they happen, small bubbles may
be removed by gentle tapping against the front glass plate, by
tapping the whole cassette onto the table or by carefully tilting
the cassette. But mind that all these measures will also disturb
your gradient.
24. To ensure having the complete pH range without disturbance
of the gradient especially in the alkaline range, you may include
pH plateaus in the front and at the end: For instance, keep
0.5 mL of the acidic and the alkaline gel solution and make the
gradient only from the rest; the acidic solution is pipetted into
the cassette before, the alkaline after the gradient.
25. The selection of acrylamido buffers or Immobilines II depends
on the pH gradient used and may vary. For detailed protocols,
see ( 17 ).