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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selected symmetrically to load the prote<strong>in</strong> samples. It is important for us to avoid<br />
confus<strong>in</strong>g the samples. At the same time, dur<strong>in</strong>g the IEF step, the weigh of the tray lid is<br />
evenly distributed over all the samples so contact between the IPGs and the electrode<br />
rema<strong>in</strong>s uniform. After all the prote<strong>in</strong> samples loaded <strong>in</strong>to the rehydration tray, the<br />
coversheet of the strips were peeled by us<strong>in</strong>g forceps. Then the strips gel side down<br />
were gently located onto the samples, respectively. And the tray was waited for an hour<br />
at the room temperature to allow to strip for soak<strong>in</strong>g the sample. After that each of the<br />
strips were overlayed with 2 - 3 ml of m<strong>in</strong>eral oil so as to hamper evaporation dur<strong>in</strong>g<br />
the rehydration process. It was added slowly, by carefully dripp<strong>in</strong>g the oil onto the<br />
plastic back<strong>in</strong>g of the strips while mov<strong>in</strong>g the pipet along the length of the strip. The<br />
f<strong>in</strong>al step of the day 1, the tray was covered with a plastic lid and lifted an <strong>in</strong>cubator<br />
(20°C) to allow to rehydrate the IPG strips.<br />
Day Two<br />
In second day, the IEF focus<strong>in</strong>g tray that has electrodes was cleaned and dried<br />
carefully. After that, paper wicks were placed at both ends of channels on the wire<br />
electrodes us<strong>in</strong>g forceps. It is crucial to prevent direct contact between IPG strips and<br />
wire electrodes, because it might cause to undesired burns on strips. Follow<strong>in</strong>g, paper<br />
wicks were wetted with 8 μl of ultrapure water <strong>in</strong> order to enable electrical conductivity.<br />
Then, strips were taken from the rehydration tray by remov<strong>in</strong>g the cover and by the help<br />
of forceps; strips were held vertically for about 5 – 10 seconds to let the m<strong>in</strong>eral oil to<br />
dra<strong>in</strong>, carefully. Then they were transferred to related channels <strong>in</strong> the focus<strong>in</strong>g tray by<br />
ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g the gel side down position. Each IPG strips were covered with 2 – 3 ml of<br />
fresh m<strong>in</strong>eral oil aga<strong>in</strong> and the lid was placed onto the focus<strong>in</strong>g tray (positive to the left<br />
when the <strong>in</strong>cl<strong>in</strong>ed portion of the tray is on the right). At the f<strong>in</strong>al step of isoelectric<br />
focus<strong>in</strong>g, the focus<strong>in</strong>g tray was put <strong>in</strong>to the PROTEAN IEF cell and the cover of the<br />
tray was closed. Isoelectric focus<strong>in</strong>g was performed at room temperature (~20°C) <strong>in</strong><br />
three steps depend<strong>in</strong>g on the values <strong>in</strong> Table 3.6.<br />
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