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

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2D-GE separates protein mixture in accordance with two distinct properties. In the first dimension, proteins are separated depending on their isoelectric point (pI, is known the pH at which a protein has no net charge) disparity, whereas in the second dimension, the system uses the molecular weight difference. It means that proteins are resolved according to their molecular weight. Isoelectric focusing (IEF) is coupling with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for protein separation, generally. Recently, the importance of 2D-GE system is rapidly increasing, as it not only can be combine with the most biological studies but also has a capacity to separate up to 10000 protein spots on one gel. Furthermore, it can monitorize more than 5000 proteins simultaneously, having nearly 2000 proteins routinely, hinging upon the pore size in acrylamide gels and pH gradient utilized. As compared to 1D-GE, it is more sensitive thanks to the properties of detection and quantification nearly 1 ng amount of protein per spot. By loading a range of protein markers whose molecular masses are already known in one of the line of the polyacrylamide gel, the molecular weights of the proteins in the sample can be estimated. Figure 2.4. Schematic Representation of 2D PAGE (Source: Nelson and Cox 2008) 33
2.1.4.2. Detection of Protein Spots and Image Analysis The last procedure of 2D-PAGE is the visualization of protein spots on gel by staining methods. Detection of protein spots can be commonly achieved by six techniques. These are Coomassie Brillant Blue (CBB) Staining, silver staining, negative staining with metal cations (e.g. zinc imidazole), staining or labeling with organic or fluorescent dyes, detection by radioactive isotopes, and by immunological detection, respectively. Among these methods, CBB staining, silver staining and fluorescence staining are the most preferred detection methods for proteomic researches. There are some notable characteristics to select the most suitable staining technique for ideal protein detection on 2D PAGE. First of all, it should be sensitive (low detection limit), well-matched with mass spectrometry and reproducible. And also it should possess linear and wide dynamic range. However, there is no method that have all of these properties exactly. Each type of protein stain has its own characteristics and limitations with regard to the sensitivity of detection and the types of proteins that stain best (Table 2.1). Gel Stain Sensitivitiy SYPRO Ruby Protein Gel Stain Literature Coomassie Brillant Blue Table 2.1. Characteristics of protein stains 1 ng Process Time and Steps 3 hr / 2 steps Advantages Mass spectrometry compatible, High detection sensitivity, High dynamic range, Reproducility, Allows protein analysis in fluorescent imagers. (Berggren et al., 2000; Nishihara and Champion, 2002; Lilley and Friedman, 2004). G-250 10 ng 2.5 hr / 3 steps R-250 40 ng 2.5hr / 2 steps R-350 40 ng 5 hr / 3 steps Mass spectrometry compatible, Easily visualized, Nonhazardous Low cost Literature (Neuhoff et al., 1988; Berggren et al., 2000; Patton, 2002; Mackintosh et al., 2003; Candiano et al., 2004). Silver Stain 1 ng 1.5hr / 3 steps High detection sensitivity, Low background Literature (Heukeshoven and Dernick, 1985; Merril et al., 1986). 34
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: is-acrylamide (N,N'-methylene-bisac
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 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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