changes in protein profiles in bortezomib applied multiple myeloma ...
changes in protein profiles in bortezomib applied multiple myeloma ...
changes in protein profiles in bortezomib applied multiple myeloma ...
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approximately 35 ml, the volume of each solution for two gels was adjusted to prepare<br />
80 ml of 12% SDS-polyacrylamide gel. Preparation of the separat<strong>in</strong>g gel and its<br />
components were described <strong>in</strong> Appendix B.<br />
After the preparation of separat<strong>in</strong>g gel components, they were mixed <strong>in</strong> a 100 ml<br />
erlenmayer flask <strong>in</strong> the order of dH2O, acrylamide / biaacrylamide, Tris-HCl, and SDS,<br />
because the polymerization began as soon as the TEMED and APS were added. Thus,<br />
they were added to the mixture at the same time and f<strong>in</strong>al mixture was poured <strong>in</strong>to the<br />
gap between glass plates without delay. After polymerization had been complete (~30 -<br />
45 m<strong>in</strong>utes), upper side of the gels were covered with tertamyl alcohol (~500 – 600 µl)<br />
and they were kept overnight at 4°C.<br />
Day Three<br />
For the equilibration, 6 ml of Equilibration buffer I was added on IPG strips<br />
which is <strong>in</strong> a clean rehydration tray with the gel side up position and the tray was<br />
carefully put on an orbital shaker for 10 m<strong>in</strong>utes. Shak<strong>in</strong>g was carried out <strong>in</strong> a slow<br />
speed to prevent the buffer from slosh<strong>in</strong>g out of the tray. At the end of the <strong>in</strong>cubation,<br />
equilibration buffer I was discarded by pour<strong>in</strong>g the liquid cautiously from the tray.<br />
Subsequent to this, 6 ml of Equilibration buffer II conta<strong>in</strong><strong>in</strong>g iodoacetamide was added<br />
on each strip and aga<strong>in</strong> tray was placed to the orbital shaker for 10 m<strong>in</strong>utes. End of the<br />
10 m<strong>in</strong>utes, equilibration buffer II was discarded as mentioned before.<br />
At the end of the equilibration of IPG strip, a graduated cyl<strong>in</strong>der (100 ml) was<br />
filled with 1X Tris - Glyc<strong>in</strong>e - SDS runn<strong>in</strong>g buffer and each bubble on the surface of the<br />
buffer was removed by the help of a Pasteur pipette. Then, the strips were sopped <strong>in</strong> the<br />
graduated cyl<strong>in</strong>der respectively to be r<strong>in</strong>sed <strong>in</strong> the buffer. Next, each strip which was<br />
positioned as gel side up was laid onto the glass plate of the SDS gel, and connected<br />
with the gel by pay<strong>in</strong>g attention not to be any air bubble at the <strong>in</strong>terface. Meanwhile, the<br />
overlay agarose solution was melted <strong>in</strong> a microwave oven by loosen<strong>in</strong>g the cap of the<br />
bottle. The glass plates were then held vertically, and the necessary amount of overlay<br />
agarose solution was poured <strong>in</strong>to the IPG well of each gel. After each strip were pushed<br />
carefully us<strong>in</strong>g a forceps, 5 m<strong>in</strong>utes was enough for agarose to be solidified. Before<br />
began to electrophoresis, the reservoir of the gel box and the gap between the gels were<br />
filled with 1X Tris - Glyc<strong>in</strong>e - SDS runn<strong>in</strong>g buffer. In order to control the cool<strong>in</strong>g of<br />
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