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

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B.5. APO LOGIX JC-1 Mitochondrial Membrane Potential Detection Kit APO LOGIX JC-1 Mitochondrial Membrane Potential Detection Kit was purchased from Cell Technology, USA. B.5.1. JC-1 Reagent The lyophilized vial was reconstituted with 500 μl DMSO to obtain a 100X stock solution. Following that, it was mixed by inverting several times at room temperature until contents were completely dissolved. Next, resuspended JC-1 reagent was aliquated in small amountssufficient for one day experimental work and stored at - 20 o C. When it was used, it was diluated 1:100 in 1X Assay Buffer (100X JC-1 reagent to 1X) immediately prior to use. B.5.2. 10X Assay Buffer 10X Assay Buffer were diluted 1:10 with ultrapure water (1 ml 10X Assay Buffer + 9 ml UP Water). B.6. 2D PAGE B.6.1. Isoelectric Focusing (IEF) B.6.1.1. Preparation of Rehydration Buffer It was used for rehydration of strips. This protocol was for including proteins in buffer. 107
Urea (8M) 4.8 g CHAPS (%2) 0.2 g DTT (50 mM) 0.077 g Ampholites (3-10) (%0.2) 100 µl Bromophenol Blue (%0.5) 1-2 µl Preparation of Rehydration Buffer Total Volume = 10 ml For urea was increasing the volume of liquid when dissolved, firstly, it was dissolved in 4ml ddH2O. Then the other chemicals were added, total solution was measured to see its volume and the final volume was bringed to 10 ml. Ampholites was diluted to 1X from the 10X stock concentration. The ampholites concentration in total volume is %0.2. Nearly, 1-2 µl Bromophenol Blue (%0.5) was used just to have color in buffer. B.6.1.2. Preparation of Equilibration Buffer I and II Stock Equilibration Buffer I (20 ml) Equilibration Buffer II (20 ml) 7.2 g / 20 ml Urea 7.2 g / 20 ml Urea 0.4 g / 20 ml SDS 0.4 g / 20 ml SDS 5 ml Ultrapura H2O 5 ml Ultrapura H2O 5 ml 1.5 M Tris – HCl pH = 8.8 5 ml 1.5 M Tris – HCl pH = 8.8 0.4 g / 20 ml DTT - 4 ml Glycerol 4 ml Glycerol - 0.5 g / 20 ml Iodoacetamide* * Iodoacetamide should be added freshly. (in Day 3). After preparation, Equilibration Buffer I was lifted to +4 o C while Equilibration Buffer II was kept at -20 o C. 108
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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
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eceptors of the Tumor Necrosis Fact
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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 and 114: A.1. RPMI-1640 Growth Medium APPEND
Page 115 and 116: Table B.1. Chemicals and Reagents U
Page 117: B.4.3. Standard Curve for BSA Table
Page 121 and 122: E. F. G. STOCK REAGENT PREPARATION
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