Biology Reference
In-Depth Information
10. Vortex secretome sample and dye to mix thoroughly, and leave
on ice in the dark for 30 min.
11. Add 1
L to
the internal standard tube to stop the reaction. Mix and leave
on ice in the dark for 10 min.
12. Snap freeze the labeled secretome samples and store at −80°C,
or proceed with IEF.
μ
L of 10 mM lysine to each Cy3/Cy5 tube and 6
μ
3.3. Immobiline
DryStrip Rehydration
and IEF
The Immobiline DryStrip gels must be rehydrated prior to IEF,
usually the night before as a minimum of 10 h is required (see
Note 12):
1.
Add 15
L of IPG buffer to 3 mL of DeStreak rehydration
solution and vortex to mix.
μ
2.
Aliquot 450
L of rehydration solution into six slots of the
re-swelling tray (see Note 13).
μ
3.
Remove the protective plastic cover from the DryStrip gels and
gently place the gels gel-side facing down into the re-swelling
tray. Ensure there are no bubbles between the gel and the tray.
4.
Cover each of the strips in each slot with 2 mL of DryStrip
cover fl uid to prevent evaporation.
5.
Replace the re-swelling tray lid and allow the strip gels to rehy-
drate overnight at room temperature (RT).
Prepare the IPGphor 3 system for sample loading and fi rst
dimension IEF.
6.
Cut electrode pads (5 × 15 mm) and wet with distilled water.
7.
Remove the DryStrip gels from the re-swelling tray using
tweezers and place them onto the ceramic manifold, position-
ing the basic end of the strip toward the negative end of the
IPGphor unit.
8.
Cover both acidic and basic ends of the strip gel with damp
electrode pads.
9.
Fasten electrodes fi rmly onto the manifold and ensure good
contact with electrode pads.
10. Clip on sample loading cups at the acidic gel end.
11. Cover the strip gels and manifold with cover fl uid, and check
for a good seal (see Note 14).
Prepare the secretome samples for loading and IEF. Each
gel should contain 25
μ
g Cy3-labeled sample, 25
μ
g Cy5-
g Cy2-labeled internal standard.
12. Combine each of the Cy3- and Cy5-labeled secretome sample
pairs as outlined in Table
1
, and add one aliquot (i.e., 1/6) of
the total volume of the Cy2 internal standard.
13. Add an equal volume of 2× sample buffer to each of the six
tubes.
labeled sample, and 25
μ
Search WWH ::
Custom Search