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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After electrophoresis was f<strong>in</strong>ished, gels were taken out from two glass plates<br />
cautiously <strong>in</strong>to a tray for sta<strong>in</strong><strong>in</strong>g process. A MALDI–TOF Mass Spectrometry<br />
compatible silver sta<strong>in</strong><strong>in</strong>g method was performed. The procedure of this mass<br />
spectrometry suitable silver sta<strong>in</strong><strong>in</strong>g technique was described <strong>in</strong> Table 3.7.<br />
3.2.9.4. Image and Data Analysis of Gel<br />
After sta<strong>in</strong><strong>in</strong>g process, gels were scanned and photographed by OLYMPUS-<br />
DP25 digital camera system. Then the image analysis, spot match<strong>in</strong>g and determ<strong>in</strong>ation<br />
of different prote<strong>in</strong> spots were performed with the 7-day free licensed software package<br />
of DECODON Delta2D Version 4.3. The software superposed the gel photos and<br />
detected the differentiated prote<strong>in</strong> spots.<br />
Accord<strong>in</strong>g to this software the differentiated prote<strong>in</strong>s were determ<strong>in</strong>ed and cut<br />
from the gel to be identified by mass spectrometry.<br />
3.2.9.5. In-Gel Digestion<br />
Differentially expressed prote<strong>in</strong> spots were <strong>in</strong>-gel digested before the mass<br />
spectrometry analysis. In 1996, Shevchenko and his co-workers def<strong>in</strong>ed a protocol that<br />
is well suited with further identification <strong>in</strong>struments(Shevchenko, et al. 1996). Protocol<br />
consists of three-day procedures which an <strong>in</strong>itial wash<strong>in</strong>g of the prote<strong>in</strong> spot and<br />
digestion reaction are performed overnight.<br />
The preparation of <strong>in</strong>-gel digestion chemicals were described <strong>in</strong> Appendix B.<br />
Before beg<strong>in</strong>n<strong>in</strong>g the <strong>in</strong>-gel digestion process, if any spots which were <strong>in</strong> a large<br />
size, they were dim<strong>in</strong>ished by the help of pipette tip.<br />
Day One: Excis<strong>in</strong>g and Desta<strong>in</strong><strong>in</strong>g of Differentiated Prote<strong>in</strong> Spots<br />
Prote<strong>in</strong> spot was excised from the gel as closely as possible with a<br />
sharp scalpel. To extend the surface area they were divided <strong>in</strong>to<br />
smaller pieces. The gel pieces were put <strong>in</strong> 1.5 ml of plastic micro<br />
centrifuge tubes, separately.<br />
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