Application of DIGE and Mass Spectrometry in the Study of Type 2 Diabetes Mellitus Mouse Models - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 317

5. It is helpful to colour-code the tops of the tubes with marker
pens which are the same colour as the dye which will be added
later. (Cy3 is red, Cy5 is blue and Cy2 is yellow). This helps to
minimise the chance of costly mix-ups at the gel preparation
stage.
6. For the 2 nmol-CyDye kit, add 5 mL of anhydrous DMF, and
for the 5 nmol-CyDye kit, add 12.5 mL of anhydrous DMF. In
order to label ten gels as described, it will be necessary to have
two of the 5 nmol- and one of the 2 nmol-CyDye kits.
7. In some cases, it is more appropriate to add the IPG buffer at
0.5% as higher concentrations can limit the maximum voltage
attainable during focusing and increase the time needed for
this step. It is added here at 2% to maximise the solubility of
the proteins.
8. If it is not convenient to process the strips directly after the
rehydration or the IEF stage, they can be frozen at −80°C.
However, they cannot be stored once the focussed strips have
been equilibrated at the stage described in Subheading 3.5 .
9. The ceramic tray must be cleaned with a detergent at neutral
pH (e.g. the proprietary cleaning solution sold for this
purpose—listed in Subheading 2.2.4 ). Other detergents could
strip off the surface of the tray, which has been treated to mini-
mise protein adsorption.
10. During rehydration, the gels swell and take on the blue colour
of the modifi ed DeStreak solution. It is thus easy to see the
ends of the (previously colourless) gels.
11. The blue colouration from the bromophenol blue (present in
the DeStreak solution) quickly clears during focusing but this
does not indicate that focusing is complete.
12. If ReadyPrep Overlay Agarose is used it can conveniently be
warmed up in the microwave oven. If many gels are being
prepared, it may be necessary to reheat the agarose to prevent
it becoming too viscous too soon.
13. It is important not to damage either gel or strip. The spatula
should only touch the backing of the IPG strip, not the gel
itself. The IPG strip is in the correct place when it is touching
the top of the second-dimension gel and there are no air bub-
bles, only agarose between the two gels.
14. The Ettan DALT twelve will hold up to 12 gel cassettes. If there
are fewer than 12 gels to be run, the vacant places must be
fi lled with blank cassette inserts. Wet the outside of each gel
cassette with the diluted run buffer in the separation unit, thus
facilitating its correct placement in the Ettan DALT twelve .