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

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The sensitivity that is achievable in staining is designated by: The amount of stain that binds to the proteins, The intensity of the coloration, The difference in coloration between stained proteins and the residual background in the body of the gel (the signal-to-noise ratio). Unbound stain molecules can be washed out of the gels without removing much stain from the proteins. Subsequent to the staining procedure, in order to analyze the spot detection, intensity, size, and shape, background correction, gel matching, normalization, quantification, etc. the gel images necessitate to be turned into digital data by using a charge-coupled device (CCD) camera or a scanner. Following, the image obtained from the CCD camera is analyzed by computer based software, such as DECODON Delta2D, PD Quest or Image J. Finally, the spots are cut out from gel and digested (in-gel digestion) into peptide fragments with trypsin enzyme to attain the determination of protein spots (newly formed, lost or up- or down-regulated) in polyacrylamide gel. Next, these spots are identified using mass spectrometry and database searches. 2.1.4.3. In-Gel Digestion In-gel digestion is the most widely used step of sample preparation for the mass spectrometric analysis of proteins. Its significance is coming from the protein databases, because they are generated depending on the peptide masses that cause peptides preferable against intact proteins. In addition to this, mass spectrometry can produce measurement errors with the increasing length of the peptide chain. The method is introduced by Rosenfeld in 1992. It can be applicable both one and two dimensional polyacrylamide gels (Rosenfeld et al., 1992). In most of the proteomic studies, trypsin is a universal choice owing to its exceptive properties as a protease for sequencing experiments with tandem mass spectrometry (MS/MS). It cleaves amide bonds in proteins at the C-terminal side of lysine and arginine residues. It forms small peptides which are generally in the mass 35
ange of 600-2500 Da. This is the ideal protein digestion for mass spectrometry. Actually, peptides having 6-20 amino acids are optimum for MS analysis and database searches. There are also different proteases exist and used for in-gel digestion process such as chymotrypsin, pepsin etc. The only difference is their cleavage characteristics. Figure 2.5. Schematic Representation of In-Gel Digestion (Source: Jiménez et al., 2001) 2.1.4.4. ZipTip (C-18 Colon) Following the in-gel digestion process, peptides are required to purify to eliminate gel contaminants such as salts, buffers, and detergents. To obtain good spectra, it is essential to keep the salt concentrations in buffers to a minimum. If the gel contaminants do not eliminate from peptides, they can interfere with mass spectrometry either inducing the poor quality mass signal or masking the peptide signals (Yates, 1998). Thus, the obtained peptide mixtures should be purified with ZipTips (Millipore). 36
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: 2.1.4.2. Detection of Protein Spots
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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