Biology Reference
In-Depth Information
3.3. DIGE Labeling
1. Prepare an experimental design whereby the biological replicates
within an experimental group are labeled equally with either
Cy3 or Cy5 (i.e., for an experiment with six biological repli-
cates per group, three should be labeled with Cy3 and three
with Cy5) (
10
). This removes a potential bias associated with
a specific dye. The internal standard, which consists of
equal amounts of protein from all samples, should be labeled
with Cy2.
2.
To prepare the internal standard, pool an appropriate volume
of each sample containing 50 mg of protein based on the results
of the protein assay. Add suffi cient Cy2 working solution such
that the dye/protein ratio is 200 pmol/100 mg (see Note 6).
3.
For the individual-labeling reactions, take the required volume
of each sample containing 100 mg of protein and add directly
to a thawed aliquot of either Cy3 and Cy5 (dye/protein ratio
of 200 pmol/100 mg).
4.
Perform the labeling reactions on ice for 30 min in darkness.
Quench with the addition of 1 mL of 10 mM lysine per
200 pmol of dye, incubating on ice for 10 min in darkness.
5.
Combine the appropriate Cy3 and Cy5 samples with 100 mg of
the Cy2-labeled internal standard and add a suffi cient amount
of TUC4 to bring the samples to equivalent volumes. Add the
required amounts of DTT solution, carrier ampholytes (IPG
buffer), and bromophenol blue such that the fi nal sample con-
sists of 10 mM DTT, 0.5% (v/v) ampholytes, and trace amounts
of bromophenol blue for color, along with 300 mg protein,
7 M urea, 2 M thiourea, 30 mM Tris-HCl, and 4% CHAPS.
6.
If sample volumes exceed the maximum allowable volume for
cup loading using the IPGphor II (150 mL), they may be con-
centrated by centrifugation using Vivaspin devices at 14,000 ×
g
at room temperature.
1.
Prior to IEF, rehydrate the 24-cm IPG strips by evenly pipetting
450 mL of rehydration solution into the groove of the reswell-
ing tray. Care should be taken to avoid introducing air bubbles.
Remove the plastic backing that protects the IPG gel, then,
without touching the gel, lay the strip down such that the gel
is facing the solution. Ensure the buffer is evenly distributed
under the strip. Add 1 mL of cover fl uid on top of each strip to
prevent evaporation of the buffer. Incubate strips overnight at
room temperature.
3.4. Two-Dimensional
Electrophoresis (2DE)
2.
Position the ceramic IPG strip tray on the IPGphor II. Align
the rehydrated IPG strips along the groove inside the manifold
with gel side facing up. If the experiment contains fewer than
12 strips, use the innermost wells fi rst leaving the outside wells
empty.
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