changes in protein profiles in bortezomib applied multiple myeloma ...

Next, 90 μl of Bradford reagent, which is composed of 5 mg CBB G-250, 2.5 ml
Absolut Ethanol and 5 ml Phosphoric Acid, were seeded in a 96-well plate (Figure 3.4),
containing 10 μl of each sample. For Blank, we used 90 μl of Bradford reagent + 10 μl
PBS. After that, the mixture in 96-well plates was allowed to incubate at room
temperature for 10 minutes. Finally, the absorbance of dye within the plates was
measured at 595 nm by Elisa reader (Thermo Electron Corporation Multiskan
Spectrum, Finland) and the standard curve was constructed by plotting the reading of
absorbance versus μg of protein in BSA standard samples. According to the equation (y
= mx+n), the best straight line was acquired. In this equation ; x, which is correspond to
protein concentration can be found with the help of y, which is the absorbance value
read at 595 nm. Dilution of the sample is required for absorbance to fall within the
linear range of the grafics.
Blank Blank Blank 50 µg/µl 50 µg/µl 50 µg/µl 1/1000
(1 nM)
1 µg/µl 1 µg/µl 1 µg/µl 1/100
(C)
1/100
(C)
1/100
(C)
1/1000
(1 nM)
2 µg/µl 2 µg/µl 2 µg/µl 1/250
(C)
1/250
(C)
1/250
(C)
1/1000
(1 nM)
4 µg/µl 4 µg/µl 4 µg/µl 1/500 1/500 1/500
(C) (C) (C)
8 µg/µl 8 µg/µl 8 µg/µl 1/1000 1/1000 1/1000
(C) (C) (C)
16
µg/µl
16
µg/µl
16
µg/µl
1/100
(1nM)
1/100
(1nM)
1/100
(1nM)
1/100
(10 nM)
20
µg/µl
20
µg/µl
20
µg/µl
1/250
(1nM)
1/250
(1nM)
1/250 1/100
(1nM) (10 nM)
40
µg/µl
40
µg/µl
40
µg/µl
1/500
(1nM)
1/500
(1nM)
1/500
(1nM)
1/100
(10 nM)
1/1000
(10 nM)
1/100
(20 nM)
1/250 (20
nM)
1/500
(20 nM)
1/1000
(10 nM)
1/100
(20 nM)
1/250
(20 nM)
1/500
(20 nM)
1/1000
(10 nM)
1/100
(20 nM)
1/250
(20 nM)
1/500
(20 nM)
1/1000
(20 nM)
1/1000
(20 nM)
Figure 3.4. Bradford Assay
1/1000
(20 nM)
1/250
(10 nM)
1/250
(10 nM)
1/250
(10 nM)
1/500
(10 nM)
1/500
(10 nM)
1/500
(10 nM)
55