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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20 nM is correspond to 20 nM Bortezomib concentration (1x10 6 U-266<br />
Cells / 2 ml / well + 20 nM Bortezomib <strong>in</strong> 2 ml),<br />
After <strong>in</strong>cubation, the cells were taken to a falcon tube and centrifugated at 1000<br />
rpm for 10 m<strong>in</strong>utes. Then, the supernatant was gently removed and discarded, while the<br />
cell pellet was lysed by add<strong>in</strong>g 50 µl of chilled Cell Lysis Buffer (1X) to get cell lysate.<br />
Then, the cell lysate was <strong>in</strong>cubated on ice for 10 m<strong>in</strong>utes before centrifugation at<br />
10000g (~14000 rpm) for 1 m<strong>in</strong>ute. Next, the supernatants were transferred to new<br />
microcentrifuge tubes. So as to measure caspase-3 enzyme activity, reaction mixture<br />
was prepared <strong>in</strong> 96-well plates with the addition of 50 µl of Reaction Buffer (2X)<br />
(conta<strong>in</strong><strong>in</strong>g 10 mM DTT), 50 µl of sample and 5 µl of caspase-3 colorimetric substrate<br />
(DEVD-pNA) and <strong>in</strong>cubated for 2 hours at 37°C <strong>in</strong> 5% CO2. After that, absorbance of<br />
the sample was read under 405 nm wavelengths by Elisa reader (Thermo Electron<br />
Corporation Multiskan Spectrum, F<strong>in</strong>land).<br />
3.2.6.2. Bradford Prote<strong>in</strong> Assay for Prote<strong>in</strong> Determ<strong>in</strong>ation<br />
Prote<strong>in</strong> concentrations were measured by Bradford assay by us<strong>in</strong>g bov<strong>in</strong>e serum<br />
album<strong>in</strong> (BSA) as the standart. The Bradford prote<strong>in</strong> assay is one of the spectroscopic<br />
analytical methods that are utilized to f<strong>in</strong>d out the total prote<strong>in</strong> concentration of a<br />
sample (Bradford 1976). The method is also known as colorimetric assay because the<br />
addition of the sample causes color <strong>changes</strong> from brown that is cationic form to blue<br />
which is anionic form. Moreover the color of the sample becomes darker and the<br />
measured absorbance rises with the prote<strong>in</strong> concentration <strong>in</strong>creases. Basic pr<strong>in</strong>ciple of<br />
this method is absorption shift from 470 nm to 595 nm. When Coomassie Brilliant Blue<br />
G-250 (CBB G-250) dye b<strong>in</strong>ds to prote<strong>in</strong>s through Van der Waals forces and<br />
hydrophobic <strong>in</strong>teractions from its sulfonic groups, absorption occurs. In general,<br />
b<strong>in</strong>d<strong>in</strong>g of dye to prote<strong>in</strong>s becomes lys<strong>in</strong>e, arg<strong>in</strong><strong>in</strong>e and histid<strong>in</strong>e residues, it also can<br />
b<strong>in</strong>d tyros<strong>in</strong>e, tryptophan and phenylalan<strong>in</strong>e weakly.<br />
Caspase-3 activity levels were normalized to prote<strong>in</strong> concentrations determ<strong>in</strong>ed<br />
by Bradford prote<strong>in</strong> assay. A series of standard prote<strong>in</strong> solutions were prepared via<br />
serial dilution us<strong>in</strong>g BSA (Bov<strong>in</strong>e Serum Album<strong>in</strong>e) diluted with 1X PBS (Phosphate<br />
Buffered Sal<strong>in</strong>e) (Table 3.1).<br />
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