Thesis final - after defense-7 - Jacobs University

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Chapter 3 3.3.2. Results and Discussion 3.3.2.1. Unfolding of proteins during HIC The 2-D gels were performed for all the chromatographic fractions collected through six different hydrophobic adsorbents and stained with colloidal blue method in order to get the precise number of spots available in each fraction. The 2-D gels have been presented in Figures 12-14 (Section 3.1) and Figures 21-23 (Section 3.2). Some denatured spots were observed for the fractions collected at high salt concentrations on Source-Phenyl and Toyopearl-Hexyl representing the conformational changes occurred during chromatography. However, Toyopearl-Butyl and Sepharose-Phenyl has revealed less conformational changes and solid spots were observed on the 2-D gels. The conformational changes at high salt concentrations during chromatography were also reported before with model proteins (122, 129-131). The ratio of the conformational changes of proteins was high on the gels collected at the high salt concentrations and extreme hydrophobic adsorbents. This revealed that conformational changes during chromatography have dependence on the salt concentration and the adsorbent type. This behavior was also reported before that by increasing the ammonium sulfate concentration and the chain length of ligands, the unfolding of protein increases on the HIC surface. This is the reason that unnecessary high salt concentration and high chain lengths of the ligands should not be preferred (129). The conformational changes observed on 2-D gels further reinforced the correlations observed in Section 3.1 and Section 3.2, between flexibility and the corresponding retention volume. Some protein loss was also observed while comparing the amount of sample applied during chromatography and proteins obtained in the fractions <strong>after</strong> chromatography. Small amount of proteins (almost 5 mg) was lost elsewhere on the hydrophobic surface. The reason could be the unfolding of the proteins which resulted in less recovery in the chromatography fractions. These results confirmed the protein unfolding during HIC with the proteome wide approach. 103
Chapter 3 3.3.2.2. Protein distributions on 2-D gels Proteins derived from S. cerevisiae cell proteome have shown less binding affinity on the adsorbents based on hydrophobic interactions. A maximum number and a high amount (mg) of proteins were eluted at the beginning of the chromatography than at the end (Figure 29 A- B). These results confirmed the less hydrophobic nature of S. cerevisiae cell proteome. The hydrophobic proteins in the S. cerevisiae cell proteome were reported to be 16% of the total cell proteome. This was very less amount than reported for other cell proteomes (104). The less hydrophobic nature of the yeast cell proteome suggested high hydrophobic conditions for the efficient separation of the cell proteome during HIC. The analysis of 2-D gels has also revealed the variation in the binding affinity of the different ligands for the same proteome. In case of Toyopearl-Ether, a higher number of protein spots were eluted at the beginning of the chromatography than at the end. The reason could be the less hydrophobic nature of the Toyopearl-Ether. In contrast, Toyopearl-Hexyl has shown higher numbers of protein spots at the end of the chromatography than at the beginning because of its high hydrophobic nature. In the similar fashion, the 2-D gels analysis has also revealed the variation in the binding affinity of the base supports for the same proteome. In case of Toyopearl-Phenyl, a higher number of proteins were eluted in the beginning of the chromatography than at the end and the reason could be the less hydrophobic nature of the base support. Sepharose-Phenyl and Source-Phenyl have shown higher numbers of spots at the end of the chromatography. This revealed the high binding affinity of the two adsorbents for the yeast cell proteome. When the adsorption affinity of polymethacrylate based media was compared with phenyl ligated media, the adsorption affinity was higher in earlier than later one. Toyopearl-Phenyl with phenyl ligand has not given higher adsorption of proteins when compared to Toyopearl- Butyl and Toyopearl-Hexyl. This confirmed that aromatic interactions have no effect on the adsorption affinity of the adsorbents (122, 132). 104
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A proteome wide approach to underst
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I also want to mention the efforts
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Dedication To my father Haji Sher A
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Table of Contents 1. Introduction .
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3.3.2.2. Protein distributions on 2
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Chapter 1 1. Introduction 1.1. Intr
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Chapter 1 cell proteome to facilita
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Chapter 1 proteins. It is this diff
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Chapter 1 Salt conc. decreasing (Gr
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Chapter 1 The ions at the start of
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Chapter 1 exposure of hydrophobic r
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Chapter 1 based on the surface hydr
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Chapter 1 1.5. Analysis of the yeas
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Chapter 1 molecular weight impuriti
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Chapter 1 followed by its introduct
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Chapter 1 protein towards purificat
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Chapter 2 EXPERIMENTAL APPROACH
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Chapter 2 Yeast cell proteome and c
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Chapter 2 ligands (Toyopearl-Hexyl,
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Chapter 2 number of spots was count
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Chapter 2 (http://www.matrixscience
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Chapter 3 RESULTS AND DISCUSSION
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Chapter 3 to the retention behavior
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Chapter 3 investigate the hydrophob
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Chapter 3 the interior of the prote
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Chapter 3 A B C Figure 9: The cryst
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Chapter 3 of the comparative analys
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Chapter 3 Figure 11: Represent the
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Chapter 3 Ether, some 2-D gels were
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Chapter 3 Figure 13: Represent 2-D
Page 61 and 62: Chapter 3 Table 5. List of the iden
Page 63 and 64: Chapter 3 Table 7. List of the iden
Page 65 and 66: Chapter 3 amino acids by Cowan-Whit
Page 67 and 68: Chapter 3 Figure 15 C: Represents t
Page 69 and 70: Chapter 3 Figure 16 A: Represents t
Page 71 and 72: Chapter 3 The polarity value for th
Page 73 and 74: Chapter 3 Figure 17: Represents the
Page 77 and 78: Chapter 3 published papers with mod
Page 79 and 80: Chapter 3 ligands revealed less dif
Page 81 and 82: Chapter 3 3.2. Influence of the dif
Page 83 and 84: Chapter 3 Table 10: The chemistry o
Page 85 and 86: Chapter 3 where approximately 50% o
Page 87 and 88: Chapter 3 3.2.3.2. The electrophore
Page 89 and 90: Chapter 3 Figure 22: Represent 2-D
Page 91 and 92: Chapter 3 Table 12. List of the ide
Page 93 and 94: Chapter 3 Table 14. List of the ide
Page 95 and 96: Chapter 3 r 2 values from the data
Page 97 and 98: Chapter 3 Figure 24 C: Represents t
Page 99 and 100: Chapter 3 approximately 47%. The me
Page 101 and 102: Chapter 3 Figure 25 B: Represents t
Page 103 and 104: Chapter 3 values have been reported
Page 105 and 106: Chapter 3 towards conformational ch
Page 109 and 110: Chapter 3 among the adsorbents were
Page 111: Chapter 3 3.3. Protein distribution
Page 115 and 116: Chapter 3 Figure 29 B: Represent th
Page 117 and 118: Chapter 3 When the number of smalle
Page 119 and 120: Chapter 3 and hydrophobic interacti
Page 121 and 122: Chapter 4 CONCLUSIONS
Page 123 and 124: Chapter 4 • The base supports rev
Page 125 and 126: Chapter 4 additional understanding
Page 127 and 128: References 13. Lienqueo, M.E.; Lese
Page 129 and 130: References 32. Salgado, J.C.; Rapap
Page 131 and 132: References 52. B.H.J., H. Accessibl
Page 133 and 134: References 71. Xindu, G. and Regnie
Page 135 and 136: References 91. Alonso-Orgaz, S.; Mo
Page 137 and 138: References 112. Gu, Z. and Glatz, C
Page 139: References 132. Reubsaet, J.L.E. an
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Thesis final - after defense-7 - Jacobs University

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