Application of 2D DIGE in Animal Proteomics - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 390

7. It is not necessary to have SDS in the separation gel if you have
an SDS-containing stacking gel.
8. Temperatures given are best to handle Indubiose A37. Other
types of agarose may need different settings. The lowest pos-
sible temperature should be selected which still ensures good
handling without solidifying the agarose too early.
9. It is good to have the standards and reference samples ready
for use in aliquots in the freezers so that you only need to thaw
and centrifuge the tubes. For the described silver staining pro-
tocol (see Subheadings 2.7 and 3.7 ), the following amounts
are recommended per gel: Take 5 mL of 1:20-diluted molecu-
lar weight markers (e.g., LMW, GE Healthcare) or 5 mL of
1:100-diluted serum, respectively, add 5 mL of sample buffer
(with freshly added DTT to 20 mg/mL), heat to 95°C for
5 min, cool by centrifugation for 5 min at 16,000 × g (e.g., on
a Centrifuge 5415C, Eppendorf), add 5 mL of 70% (v/v) glyc-
erol and mix.
10. Alternatively, standards may be applied on fi lter application
pieces which are put directly on the fl at surface of the stacking
gel, side by side to the IPG strip. Take care that no proteins
leak out of the fi lter and into the space reserved for the IPG
strip, thus contaminating the sample.
11. Staining is best performed in glass vessels. Except for the devel-
oper, which is needed for more than one bath, 200-250 mL of
each solution are enough for this gel size.
12. Good quality of the sodium carbonate is essential to keep back-
ground staining low.
13. Styrofoam boxes with lids serve well for ice baths and keeping
samples in the dark.
14. The pH of the protein sample dissolved or diluted in labeling
buffer should be checked, at least for each sample type and/or
condition and, if necessary, readjusted. This is of special impor-
tance if samples have been TCA-precipitated.
15. Before starting the incubation, make sure that the components
are thoroughly mixed and that all liquid is at the bottom of the
tubes. Small droplets of sample or dye elsewhere on the walls of
the tubes are brought down by spinning the samples briefl y at
high speed. Pipetting of such small volumes is not easy. Wiping
the tips on the wall of the dye tube helps to have more uniform
dye amounts in the samples. This ensures comparable sample
labeling and facilitates the selection of optimal scanning condi-
tions for each gel. There are special pipettes which allow measur-
ing such small volumes, e.g., Research 0.1-2.5 mL (Eppendorf).
16. Labeled single samples are kept apart and are combined only
shortly before sample application, to avoid cross-reactions.