Application of 2D DIGE in Animal Proteomics - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 394

the protruding fi lm of the cathodic side. The strips should fl oat
in the solution and not stick to the glass tube.
41. If the plastic support protrudes less than 5 mm, the strip should
fi t nicely into the trough in the stacking gel. Otherwise you
need to shorten it.
42. The anodic running buffer may be used up to 5-10 times, the
cathodic buffer only once.
43. Saturation of spots is easily noticed during scanning, as these
spots appear red. When the run is completed and the software
ImageQuant opens immediately, the different channels of the
scan may be split (click on the appropriate buttons) and the
cursor moved slowly over the albumin spot. If “100004.25
Counts” appears, the spot is saturated, and for the next scan less
volts PMT should be chosen. If on the other hand the value is
below 40,000-50,000, PMT settings should be increased.
44. If gels cannot be scanned immediately or if you want to re-scan
them at a later time, storage in fi xing solution is possible. Although
without the support of the glass plates the shape of the gels will
change slightly (due to their swelling in the solution), fl uores-
cence is preserved for several days/weeks at least for qualitative
purposes and when gels are kept in a (dark) cold room.
45. If you intend to include mass spectrometric identifi cation of
protein spots in your workfl ow, you have to avoid introducing
contaminant proteins (e.g., keratins). This means special
emphasis on purity of all labware and solutions, wearing gloves,
etc.
46. Fixing solution is also a good choice for storing 2DE gels, for
instance while waiting for the results of your quantifi cation. In
this solution, gels may be kept for days, even weeks. However,
there may be changes in the patterns, e.g., loss of small mole-
cules for which the fi xing solution is not “strong” enough to
denature and immobilize them in the gel. This will need to be
checked from case to case.
47. Solutions containing silver ions should be collected for separate
disposal. This can best be done by combining them with the fi rst
wash of developer. In this alkaline solution, silver precipitates as
hydroxide and may be collected in a concentrated form.
48. It is not always possible to fi nd DIGE-detected spots in silver-
stained gels, especially spots of smaller proteins. This is due to
the different staining properties of the dyes/stains used ( 7 ).
49. Silver-stained gels can be kept at 4-6°C in water for days/
weeks before cutting spots for MS analysis. However, usually
best results are obtained with “fresh” spots.