Biology Reference
In-Depth Information
3. Mix the dye and protein sample thoroughly by vortexing.
4. Centrifuge briefl y in a microcentrifuge to collect the solution
at the bottom of the tube. Leave the tube on ice and in the
dark for 30 min.
5. Add 1 mL of 10 mM lysine to stop the reaction. Mix and spin
the tube briefl y in a microcentrifuge. Leave the tube on ice and
in the dark for 10 min.
Labeling is now fi nished. The labeled samples can be processed
immediately or stored for up to 3 months at −80°C in the dark.
The main difference between conventional 2D gel electrophoresis
and DIGE is that the latter will enable up to three different protein
samples to be run on a single 2D gel. To achieve this, you need to
mix the differentially labeled protein samples before the fi rst dimen-
sion run.
3.7. Isoelectric
Focusing
1. Rehydrate the Immobiline DryStrips for 8-22 h (gel side
down), without the protein sample, in the Immobiline DryStrip
reswelling tray. The total volume applied to the strip depends
on its length and the manufacturer's instructions. We used
18 cm pH 3-11 NL Immobiline DryStrip, a total volume of
340 mL of rehydration buffer containing 0.5% ampholytes and
1.2% DeStreak per strip. The strip is then covered with 2 mL
of IPG cover fl uid.
2. After rehydration of the Immobiline DryStrip, hold the end of
the strips with forceps and lift the strip out of the tray. Allow
excess cover fl uid to run off the Immobiline DryStrip onto a
piece of tissue. Place the Immobiline DryStrip gel side up with
the basic end (fl at end of Immobiline DryStrip) and fl ush with
the fl at end of the IPGphor cup-loading strip holder.
3. Prepare electrode pads by cutting 5 × 10 mm pieces from the
IEF electrodes strips. Place these on a clean dry surface such as
a plate and soak with distilled water. Remove excess water by
blotting with fi lter paper. Place a prepared damp electrode pad
onto the acidic and basic end of the gel.
4. Clip down the electrodes fi rmly onto the electrode pad. Ensure
that there is a good contact with the Immobiline DryStrip and
the metal on the outside of the strip holder.
5. Apply at least 4 mL of DryStrip cover fl uid to the IPGphor
cup-loading strip holder, allowing the oil to spread so it com-
pletely covers the Immobiline DryStrip.
6. Clip the loading cup in the acidic end of the strip, so it is posi-
tioned between the two electrodes.
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