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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Next, 90 μl of Bradford reagent, which is composed of 5 mg CBB G-250, 2.5 ml<br />
Absolut Ethanol and 5 ml Phosphoric Acid, were seeded <strong>in</strong> a 96-well plate (Figure 3.4),<br />
conta<strong>in</strong><strong>in</strong>g 10 μl of each sample. For Blank, we used 90 μl of Bradford reagent + 10 μl<br />
PBS. After that, the mixture <strong>in</strong> 96-well plates was allowed to <strong>in</strong>cubate at room<br />
temperature for 10 m<strong>in</strong>utes. F<strong>in</strong>ally, the absorbance of dye with<strong>in</strong> the plates was<br />
measured at 595 nm by Elisa reader (Thermo Electron Corporation Multiskan<br />
Spectrum, F<strong>in</strong>land) and the standard curve was constructed by plott<strong>in</strong>g the read<strong>in</strong>g of<br />
absorbance versus μg of prote<strong>in</strong> <strong>in</strong> BSA standard samples. Accord<strong>in</strong>g to the equation (y<br />
= mx+n), the best straight l<strong>in</strong>e was acquired. In this equation ; x, which is correspond to<br />
prote<strong>in</strong> concentration can be found with the help of y, which is the absorbance value<br />
read at 595 nm. Dilution of the sample is required for absorbance to fall with<strong>in</strong> the<br />
l<strong>in</strong>ear range of the grafics.<br />
Blank Blank Blank 50 µg/µl 50 µg/µl 50 µg/µl 1/1000<br />
(1 nM)<br />
1 µg/µl 1 µg/µl 1 µg/µl 1/100<br />
(C)<br />
1/100<br />
(C)<br />
1/100<br />
(C)<br />
1/1000<br />
(1 nM)<br />
2 µg/µl 2 µg/µl 2 µg/µl 1/250<br />
(C)<br />
1/250<br />
(C)<br />
1/250<br />
(C)<br />
1/1000<br />
(1 nM)<br />
4 µg/µl 4 µg/µl 4 µg/µl 1/500 1/500 1/500<br />
(C) (C) (C)<br />
8 µg/µl 8 µg/µl 8 µg/µl 1/1000 1/1000 1/1000<br />
(C) (C) (C)<br />
16<br />
µg/µl<br />
16<br />
µg/µl<br />
16<br />
µg/µl<br />
1/100<br />
(1nM)<br />
1/100<br />
(1nM)<br />
1/100<br />
(1nM)<br />
1/100<br />
(10 nM)<br />
20<br />
µg/µl<br />
20<br />
µg/µl<br />
20<br />
µg/µl<br />
1/250<br />
(1nM)<br />
1/250<br />
(1nM)<br />
1/250 1/100<br />
(1nM) (10 nM)<br />
40<br />
µg/µl<br />
40<br />
µg/µl<br />
40<br />
µg/µl<br />
1/500<br />
(1nM)<br />
1/500<br />
(1nM)<br />
1/500<br />
(1nM)<br />
1/100<br />
(10 nM)<br />
1/1000<br />
(10 nM)<br />
1/100<br />
(20 nM)<br />
1/250 (20<br />
nM)<br />
1/500<br />
(20 nM)<br />
1/1000<br />
(10 nM)<br />
1/100<br />
(20 nM)<br />
1/250<br />
(20 nM)<br />
1/500<br />
(20 nM)<br />
1/1000<br />
(10 nM)<br />
1/100<br />
(20 nM)<br />
1/250<br />
(20 nM)<br />
1/500<br />
(20 nM)<br />
1/1000<br />
(20 nM)<br />
1/1000<br />
(20 nM)<br />
Figure 3.4. Bradford Assay<br />
1/1000<br />
(20 nM)<br />
1/250<br />
(10 nM)<br />
1/250<br />
(10 nM)<br />
1/250<br />
(10 nM)<br />
1/500<br />
(10 nM)<br />
1/500<br />
(10 nM)<br />
1/500<br />
(10 nM)<br />
55