changes in protein profiles in bortezomib applied multiple myeloma ...

approximately 35 ml, the volume of each solution for two gels was adjusted to prepare
80 ml of 12% SDS-polyacrylamide gel. Preparation of the separating gel and its
components were described in Appendix B.
After the preparation of separating gel components, they were mixed in a 100 ml
erlenmayer flask in the order of dH2O, acrylamide / biaacrylamide, Tris-HCl, and SDS,
because the polymerization began as soon as the TEMED and APS were added. Thus,
they were added to the mixture at the same time and final mixture was poured into the
gap between glass plates without delay. After polymerization had been complete (~30 -
45 minutes), upper side of the gels were covered with tertamyl alcohol (~500 – 600 µl)
and they were kept overnight at 4°C.
Day Three
For the equilibration, 6 ml of Equilibration buffer I was added on IPG strips
which is in a clean rehydration tray with the gel side up position and the tray was
carefully put on an orbital shaker for 10 minutes. Shaking was carried out in a slow
speed to prevent the buffer from sloshing out of the tray. At the end of the incubation,
equilibration buffer I was discarded by pouring the liquid cautiously from the tray.
Subsequent to this, 6 ml of Equilibration buffer II containing iodoacetamide was added
on each strip and again tray was placed to the orbital shaker for 10 minutes. End of the
10 minutes, equilibration buffer II was discarded as mentioned before.
At the end of the equilibration of IPG strip, a graduated cylinder (100 ml) was
filled with 1X Tris - Glycine - SDS running buffer and each bubble on the surface of the
buffer was removed by the help of a Pasteur pipette. Then, the strips were sopped in the
graduated cylinder respectively to be rinsed in the buffer. Next, each strip which was
positioned as gel side up was laid onto the glass plate of the SDS gel, and connected
with the gel by paying attention not to be any air bubble at the interface. Meanwhile, the
overlay agarose solution was melted in a microwave oven by loosening the cap of the
bottle. The glass plates were then held vertically, and the necessary amount of overlay
agarose solution was poured into the IPG well of each gel. After each strip were pushed
carefully using a forceps, 5 minutes was enough for agarose to be solidified. Before
began to electrophoresis, the reservoir of the gel box and the gap between the gels were
filled with 1X Tris - Glycine - SDS running buffer. In order to control the cooling of
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