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

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APPENDIX B CHEMICALS, REAGENTS AND SOLUTIONS B.1. Bortezomib Bortezomib was obtained from LCLabs (USA). The stock solution of Bortezomib was prepared in dimethyl sulfoxide (DMSO) at a concentration of 2,6 mmol/ml (1,5 mg of Bortezomib was dissolved in 1 ml DMSO), stored at -20 o C and diluted in cell culture medium. Molecular weight of Bortezomib is 384.24 g/mol. B.2. MM U-266 Cells U-266 human MM cells were kindly obtained from the German Collection of Microorganisms and Cell Cultures (Germany). Table B.1. Chemicals and Reagents Used in Experiments NO CHEMICALS COMPANY 1 Dimethyl Sulfoxide (DMSO) Sigma 2 Trypan Blue Dye Sigma 3 Phosphate Buffered Saline (PBS) Invitrogen 4 Penicillin-Streptomycin Invitrogen 5 Fetal Bovine Serum (FBS) Invitrogen 6* XTT Reagent (should not be exposed to light) 7* Activation Reagent (should not be exposed to light) 8** DEVD-pNA (should not be exposed to light) (Stored at -20 o C) 9** Cell Lysis Buffer (Stored at +4 o C) 10** 2X Reaction Buffer (Stored at -20 o C) 11** Dithiothreitol (DTT) (Stored at -20 o C) Biological Industries Biological Industries BioVision BioVision BioVision BioVision cont. on next page 103
Table B.1. Chemicals and Reagents Used in Experiments (cont.) 12 Bovine Serum Albumine (BSA) Sigma 13 Coomassie Brilliant Blue G-250 (CBB G-250) Dye AppliChem 14 Absolute Ethanol AppliChem 15 Phosphoric Acid AppliChem 16*** JC-1 Reagent 5,5’,6,6’-tetrachloro-1,1’,3,3’tetraethylbenzimidazolylcarbocyanine iodide (lyophilized) Cell Technology 17*** 10X Assay Buffer Cell Technology 18 Urea (8M) AppliChem 19 Thiourea AppliChem 20 Glycerol AppliChem 21 Ampholites (3-10) (%0.2) Fluka 22 Bromophenol Blue (%0.5) AppliChem 23 CHAPS (%2) AppliChem 24 Agarose AppliChem 25 SDS AppliChem 26 phosphoric acid AppliChem 27 Iodoacetamide Sigma 28 Acrylamide AppliChem 29 Bisacrylamide AppliChem 30 1.5 M Tris – HCl pH = 8.8 AppliChem 31 Ammoniun Persulfate (APS) Sigma 32 Tetramethylethylenediamine (TEMED) Sigma 6,7* are XTT Cell Proliferation Kit Components, 8, 9, 10, 11** are Caspase-3 Colorimetric Assay Kit Components, 16, 17*** are APO LOGIX JC-1 Mitochondrial Membrane Potential Detection Kit Components. B.3. XTT Cell Proliferation Assay Kit Israel. XTT Cell Proliferation Assay Kit was obtained from Biological Industries, 104
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CHANGES IN PROTEIN PROFILES IN BORT
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ACKNOWLEDGEMENTS First of all, with
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ÖZET BORTEZOMĠB UYGULANAN MULTĠP
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2.1.4.1.2. Two-Dimensional Gel Elec
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LIST OF FIGURES Figure Page Figure
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LIST OF TABLES Table Page Table 1.1
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When normal cells in the body begin
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Figure 1.2. Development of Cancer S
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the myeloma cells does not help fig
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A rare form of MM called Nonsecreto
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1.2.3. Risk Factors for MM A risk f
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of the mineralizations by adjusting
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To measure the levels of red cells,
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The levels of blood urea nitrogen (
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By using the system introduced by D
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1.2.7.2. Biology of The Proteasome
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Figure 1.10. Ubiquitin-Proteasome P
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Figure 1.12. Effect of Bortezomib o
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encompasses the investigation of pr
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of modification. Ubiquitin is a sma
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the given cells. On the other hand,
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is-acrylamide (N,N'-methylene-bisac
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2.1.4.2. Detection of Protein Spots
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ange of 600-2500 Da. This is the id
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ions resolved by the mass analyzer
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2.1.4.5.2. Mass Analyzers Figure 2.
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There is no doubt that different ma
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mask low-quantity proteins that may
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3.1. Materials 3.1.1. Medias CHAPTE
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3.2.5. Cell Proliferation Assay Ant
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After 72 hour incubation period at
Page 63 and 64: eceptors of the Tumor Necrosis Fact
Page 65 and 66: The preparation of stock BSA Standa
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: A.1. RPMI-1640 Growth Medium APPEND
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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