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 Cells / 2 ml / well + 20 nM Bortezomib in 2 ml), After incubation, the cells were taken to a falcon tube and centrifugated at 1000 rpm for 10 minutes. Then, the supernatant was gently removed and discarded, while the cell pellet was lysed by adding 50 µl of chilled Cell Lysis Buffer (1X) to get cell lysate. Then, the cell lysate was incubated on ice for 10 minutes before centrifugation at 10000g (~14000 rpm) for 1 minute. Next, the supernatants were transferred to new microcentrifuge tubes. So as to measure caspase-3 enzyme activity, reaction mixture was prepared in 96-well plates with the addition of 50 µl of Reaction Buffer (2X) (containing 10 mM DTT), 50 µl of sample and 5 µl of caspase-3 colorimetric substrate (DEVD-pNA) and incubated for 2 hours at 37°C in 5% CO2. After that, absorbance of the sample was read under 405 nm wavelengths by Elisa reader (Thermo Electron Corporation Multiskan Spectrum, Finland). 3.2.6.2. Bradford Protein Assay for Protein Determination Protein concentrations were measured by Bradford assay by using bovine serum albumin (BSA) as the standart. The Bradford protein assay is one of the spectroscopic analytical methods that are utilized to find out the total protein concentration of a sample (Bradford 1976). The method is also known as colorimetric assay because the addition of the sample causes color changes from brown that is cationic form to blue which is anionic form. Moreover the color of the sample becomes darker and the measured absorbance rises with the protein concentration increases. Basic principle of this method is absorption shift from 470 nm to 595 nm. When Coomassie Brilliant Blue G-250 (CBB G-250) dye binds to proteins through Van der Waals forces and hydrophobic interactions from its sulfonic groups, absorption occurs. In general, binding of dye to proteins becomes lysine, arginine and histidine residues, it also can bind tyrosine, tryptophan and phenylalanine weakly. Caspase-3 activity levels were normalized to protein concentrations determined by Bradford protein assay. A series of standard protein solutions were prepared via serial dilution using BSA (Bovine Serum Albumine) diluted with 1X PBS (Phosphate Buffered Saline) (Table 3.1). 53
The preparation of stock BSA Standard was given in Appendix B. Table 3.1. Preparation of BSA Standards and Test Sample for the Bradford Protein Assay Serial Dilution Test Sample BSA Standards Volume, µl PBS Volume, µl Blank - 10 50 µg/µl 200 200 40 µg/µl 160 40 20 µg/µl 100 100 16 µg/µl 80 20 8 µg/µl 50 50 4 µg/µl 50 50 2 µg/µl 50 50 1 µg/µl 50 50 After the preparation of standard protein (BSA), protein samples were diluted by the ratios 1:100, 1:250, 1:500 and 1:1000) (Table 3.2). C 1 nM 10 nM 20 nM Table 3.2. Dilution of Protein Sample 1/100 10 µl Sample from C + 90 µl PBS 1/250 20 µl Sample from C + 480 µl PBS 1/500 20 µl Sample from C + 980 µl PBS 1/1000 10 µl Sample from C + 990 µl PBS 1/100 10 µl Sample from 1 nM + 90 µl PBS 1/250 20 µl Sample from 1 nM + 480 µl PBS 1/500 20 µl Sample from 1 nM + 980 µl PBS 1/1000 10 µl Sample from 1 nM + 990 µl PBS 1/100 10 µl Sample from 10 nM + 90 µl PBS 1/250 20 µl Sample from 10 nM + 480 µl PBS 1/500 20 µl Sample from 10 nM + 980 µl PBS 1/1000 10 µl Sample from 10 nM + 990 µl PBS 1/100 10 µl Sample from 20 nM + 90 µl PBS 1/250 20 µl Sample from 20 nM + 480 µl PBS 1/500 20 µl Sample from 20 nM + 980 µl PBS 1/1000 10 µl Sample from 20 nM + 990 µl PBS 54
Page 1 and 2:
CHANGES IN PROTEIN PROFILES IN BORT
Page 3 and 4:
ACKNOWLEDGEMENTS First of all, with
Page 5 and 6:
ÖZET BORTEZOMĠB UYGULANAN MULTĠP
Page 7 and 8:
2.1.4.1.2. Two-Dimensional Gel Elec
Page 9 and 10:
LIST OF FIGURES Figure Page Figure
Page 11 and 12:
LIST OF TABLES Table Page Table 1.1
Page 13 and 14: When normal cells in the body begin
Page 15 and 16: Figure 1.2. Development of Cancer S
Page 17 and 18: the myeloma cells does not help fig
Page 19 and 20: A rare form of MM called Nonsecreto
Page 21 and 22: 1.2.3. Risk Factors for MM A risk f
Page 23 and 24: of the mineralizations by adjusting
Page 25 and 26: To measure the levels of red cells,
Page 27 and 28: The levels of blood urea nitrogen (
Page 29 and 30: By using the system introduced by D
Page 31 and 32: 1.2.7.2. Biology of The Proteasome
Page 33 and 34: Figure 1.10. Ubiquitin-Proteasome P
Page 35 and 36: Figure 1.12. Effect of Bortezomib o
Page 37 and 38: encompasses the investigation of pr
Page 39 and 40: of modification. Ubiquitin is a sma
Page 41 and 42: the given cells. On the other hand,
Page 43 and 44: is-acrylamide (N,N'-methylene-bisac
Page 45 and 46: 2.1.4.2. Detection of Protein Spots
Page 47 and 48: ange of 600-2500 Da. This is the id
Page 49 and 50: ions resolved by the mass analyzer
Page 51 and 52: 2.1.4.5.2. Mass Analyzers Figure 2.
Page 53 and 54: There is no doubt that different ma
Page 55 and 56: mask low-quantity proteins that may
Page 57 and 58: 3.1. Materials 3.1.1. Medias CHAPTE
Page 59 and 60: 3.2.5. Cell Proliferation Assay Ant
Page 61 and 62: After 72 hour incubation period at
Page 63: eceptors of the Tumor Necrosis Fact
Page 67 and 68: 3.2.6.3. Detection of the Loss of M
Page 69 and 70: centrifugation, the supernatant was
Page 71 and 72: selected symmetrically to load the
Page 73 and 74: approximately 35 ml, the volume of
Page 75 and 76: After electrophoresis was finished,
Page 77 and 78: 7 Day Two: Reduction, Alkylation, a
Page 79 and 80: CHAPTER 4 RESULTS AND DISCUSSION 4.
Page 81 and 82: % Changes in Cytoplasmic/Mitochondr
Page 83 and 84: Table 4.1. Magnified Segments of Di
Page 85 and 86: Table 4.1. Magnified Segments of Di
Page 87 and 88: After labeling, black and red color
Page 89 and 90: 78 7 8 Y2S Y3S Table 4.2. Results o
Page 91 and 92: 80 12 S4Ca Table 4.2. Results of Di
Page 93 and 94: 82 27 Y5S Table 4.2. Results of Dif
Page 95 and 96: Spot 4, Ubiquitin-conjugating enzym
Page 97 and 98: is a transcriptional repressor, but
Page 99 and 100: activity and loss of mitochondrial
Page 101 and 102: distributed, but correlate with nat
Page 103 and 104: DeMartino, G. N. and Slaughter, C.
Page 105 and 106: proteomic analyses of a systematica
Page 107 and 108: Mahindraa, A., Hideshimab, T. and A
Page 109 and 110: Richardson, P. G., Hideshima, T. an
Page 111 and 112: Terpos, E. 2008. Bortezomib Directl
Page 113 and 114: A.1. RPMI-1640 Growth Medium APPEND
Page 115 and 116:
Table B.1. Chemicals and Reagents U
Page 117 and 118:
B.4.3. Standard Curve for BSA Table
Page 119 and 120:
Urea (8M) 4.8 g CHAPS (%2) 0.2 g DT
Page 121 and 122:
E. F. G. STOCK REAGENT PREPARATION
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