Urinary Proteome Profiling Using 2D-DIGE and LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 298

specific and should be increased for higher protein loads
( see Note 8 ). Apparatus is covered to exclude light.

(1) Sets of clean low-fluorescence glass plates containing large
back plate (with spacer attached) and smaller 'front plates'
(Ettan DALT 24 cm gel plates) are selected depending on
the number of gels that needs to be run. Spacer size can be
varied for 0.5, 1.0 and 1.5 mm gel thickness.
(2) Reference markers are applied to the surface of the smaller
plates. These are placed halfway down the plates and
15-20 mm from each edge and are critical for determin-
ing coordinates for automated spot picking. 1.5 ml of fresh
Bind Silane solution is applied per small plate and surface
wiped with lint-free tissue. Plates are left to dry for a mini-
mum of 1 h. The inner surface of the larger spacer plate is
treated with Repel Silane, wiped using lint-free tissues and
left to dry for 10 min.
(3) Glass plates with the repel and bind surfaces facing each
other are assembled in Ettan gel casting unit assuring a
tight seal and no leak. Feeding tube and funnel are attached
to the caster and the required polyacrylamide gel solution
is poured ensuring no air bubbles are introduced. Water-
saturated butanol (2 ml) is overlaid on each gel and gels are
allowed to polymerise for at least an hour ( see Note 9 ).
(4) On completion of the first dimension separation, gel strips
are equilibrated for 15 min in equilibration buffer contain-
ing 65 mM DTT and then 15 min in the same buffer con-
taining 240 mM IAM ( see Note 10 ).
(5) The equilibrated Immobiline gel strips are then rinsed with
1X SDS electrophoresis buffer prior to being placed onto
the top of handmade PAGE gels described in Section 3.5 .
Strips are placed in a molten 0.5% agarose overlay, with the
basic end of the strip towards the left-hand side with the
bonded plate facing forward ( see Note 11 ).
(6) The agarose is allowed to cool and set at room temperature.
(7) The DALT twelve electrophoresis tank is filled with SDS
running buffer and gels complete with Immobiline Drys-
trips are inserted into the designated slots in the gel tank.
Unoccupied slots are filled with plastic blanks and top tank
filled with running buffer.
(8) The Ettan DALT twelve system is run for 16 h at 2.2 W per
gel or until the dye front had reached the bottom of the gel
( see Note 12 ).

3.3.3.Second
DimensionGel
Preparation
andSeparation

(1) Gels (still between glass plates) are rinsed in Milli-Q 18

water prior to image capture ( see Note 13 ). Gels plates

3.3.4. ImageCapture