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

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electrophoresis, water circulation was used and electrophoresis was started at constant current at 32 mA for 30 minutes and followed by 50 mA for 5 – 6 hours. To monitor the progress of the electrophoresis, Bromophenol Blue was used. When it reached the bottom of the gel, electrophoresis was stopped. 3.2.9.3. Staining of the Gel A number of staining techniques have been developed to stain polypeptides with silver salts after separation by two-dimensional gel electrophoresis. In every case, the procedure based on differential reduction of silver ions that are bound to the side chains of amino acids (Switzer et al., 1979; Nielsen and Brown, 1984; Merril et al., 1984). 1 Table 3.7. Time Coarse for MALDI-TOF Compatible Mass Spectrometry (Source: Adapted from He et al., 2003; He et al., 2004; Lin et al., 2008) STEP SOLUTION Fixing (Fixer Soln.) 40% EtOH, 10% HAc; TIME OF TREATMENT > 1 hour (overnight) COMMENTS Overnight incubation is all right 2 Washing - Pretreatment Soln. - - 3 Sensitizing 30% EtOH, 4.1% NaAc, 0.2% Na2S2O3; 30 minutes Time should be exact 4 Washing dH2O 5 min. x 3 2 nd & 3 rd Silver Nitrate Solution solution should always be fresh 5 Impregnate 0.1% AgNO3, 0.02% formalin; 20 min. - 6 7 Washing Developing 8 Stop dH2O Developing Solution 2.5% Na2CO3, 0.01% formalin Stop Solution 1.46% EDTA 1 min. x 2 ~ 10 min. > 10 minutes 9 Washing dH2O 5 min. x 3 Time should be exact 2 nd & 3 rd solution should always be fresh After a few minutes, add some dH2O to proceed the reaction slowly. Time should be determined by observation of color development The gels can be kept in this solution overnight 2 nd & 3 rd solution should always be fresh 63
After electrophoresis was finished, gels were taken out from two glass plates cautiously into a tray for staining process. A MALDI–TOF Mass Spectrometry compatible silver staining method was performed. The procedure of this mass spectrometry suitable silver staining technique was described in Table 3.7. 3.2.9.4. Image and Data Analysis of Gel After staining process, gels were scanned and photographed by OLYMPUS- DP25 digital camera system. Then the image analysis, spot matching and determination of different protein spots were performed with the 7-day free licensed software package of DECODON Delta2D Version 4.3. The software superposed the gel photos and detected the differentiated protein spots. According to this software the differentiated proteins were determined and cut from the gel to be identified by mass spectrometry. 3.2.9.5. In-Gel Digestion Differentially expressed protein spots were in-gel digested before the mass spectrometry analysis. In 1996, Shevchenko and his co-workers defined a protocol that is well suited with further identification instruments(Shevchenko, et al. 1996). Protocol consists of three-day procedures which an initial washing of the protein spot and digestion reaction are performed overnight. The preparation of in-gel digestion chemicals were described in Appendix B. Before beginning the in-gel digestion process, if any spots which were in a large size, they were diminished by the help of pipette tip. Day One: Excising and Destaining of Differentiated Protein Spots Protein spot was excised from the gel as closely as possible with a sharp scalpel. To extend the surface area they were divided into smaller pieces. The gel pieces were put in 1.5 ml of plastic micro centrifuge tubes, separately. 64
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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
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 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: approximately 35 ml, the volume of
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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