Biology Reference
In-Depth Information
are available from different companies (Bio-Rad, GE
Healthcare, Sigma, etc.). Most of these methods are based on
a short protocol of precipitation before the quantifi cation by
colorimetry. The optimal concentration for the labelling reac-
tion is around 5
μ
g/
μ
L.
5. 30
g of protein is labelled for each sample by Cy3 and Cy5,
and the internal standard is prepared with an equal part of each
sample, before the labelling with Cy2, classically used for the
internal standard (
see
Note 5
).
6. Each CyDye is added to the Eppendorf tube containing the pro-
tein solution. The ratio of 400 pmol/
μ
g of pro-
tein has to be respected to optimize the labelling reaction, i.e., in
our laboratory, 0.24
μ
L of dye to 50
μ
μ
L of dye solution is added to each tube
containing 30
g of protein solution. After vortexing and a short
centrifugation to concentrate the solution at the bottom of the
tube, the mixture is incubated for 30 min on ice in the dark.
μ
7. After 30 min of incubation, 1
L of stopping solution is added
and the mixture is vortexed and incubated again for 10 min on
ice in the dark.
8. The labelled samples can be stored at −80 °C (for up to
3 months) or be used directly (
see
Note 6
).
μ
1. The labelled samples have to be combined in one tube before
the loading; each gel should contain 30
3.3 First-Dimension
Separation:
Isoelectrofocusing
μ
g of protein labelled
with Cy3, a second sample of 30
μ
g labelled with Cy5, and
g of internal standard labelled with Cy2.
2. The volume of each combination has to be set at 120
30
μ
L before
cup loading by adding 2× sample buffer solution containing
DTT and ampholytes.
3. The rehydrated IPG strips are removed from the reswelling
tray and transferred, gel side up, into the manifold properly
positioned on the IPGphor system.
4. Electrode paper wicks, wetted with deionized water, are placed
at the end of the IPG strips. Electrode is set on top of these
papers before the positioning of the cup. It is important that
the electrode wires are in contact with the paper.
5. IPG strips have to be covered with mineral oil to avoid drying
during the fi rst-dimension run. Pour around 100 mL on top of
the gels into the manifold (
see
Note 7
).
6. The combination of the three samples (labelled with Cy3, Cy5,
and internal standard) is loaded in the cup. Before starting the
electrophoretic run, the samples have to be covered with min-
eral oil, still to avoid desiccation and the formation of urea
crystals in the cups.
7. The electrophoretic run can be started: in this example, the
conditions of the run are the following at 20 °C: 100 V for 2 h,
μ
