Thesis final - after defense-7 - Jacobs University

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Chapter 3 work, the effects of larger proteins (> 50 KDa) on retention have been examined as a function of the adsorbent type. The number of larger proteins in each fraction was divided by the total number of proteins to normalize the data. The normalized values of the larger proteins from the three ligands was presented in Figure 30, where the larger proteins were found to be higher at low salt concentrations than at high salt concentrations. In other words, the larger proteins were higher in the latter fractions in comparison to the earlier ones. This revealed that larger proteins retained longer on the adsorbents. In case of larger proteins, the retention was higher for Toyopearl-Butyl and Toyopearl-Hexyl in comparison to the other adsorbents. It was reported before that if the pore space of the adsorbent is smaller than the dimensions of the magnitude as the protein has, then it will result in less retention of protein (33). However, this was not applicable on Toyopearl-Ether and Toyopearl-Phenyl adsorbents where larger proteins has shown less retention, although they have high pore size (100 nm). The reason could be the less hydrophobic nature of the two adsorbents which retained larger proteins for a shorter time during chromatography. In a similar way, Source-Phenyl has given less retention to larger proteins, although it has a high hydrophobic nature, but the pore size is smaller (34 nm). These results declared that the pore size and hydrophobicity of the adsorbents could be the combine contributing parameters affecting the retention of larger proteins. In case of smaller proteins it was reported once that it has no effect on retention (33). However, in another report it was reported that smaller proteins exhibited a decrease in retention due to the less contact surface area with adsorbents (68). This was confirmed in this work when smaller proteins have exhibited an inverse relationship to the corresponding retention behavior and most of the smaller proteins were eluted at the beginning of the chromatography. The reason could be that smaller proteins have less non-polar surface area to interact with the adsorbents. 107
Chapter 3 When the number of smaller and larger proteins was compared, smaller proteins were three times higher than larger proteins on the 2-D gels. This revealed that S. cerevisiae cell proteome have a higher proportion of smaller proteins than larger proteins. Figure 30: Represents the normalized values of the larger proteins (Mr > 50) in each fraction as a function of the different ligands. The larger proteins revealed a significant correlation with the respective salt concentrations where the fractions were collected. 3.3.2.3. The influence of protein isoelectric point on the chromatographic behavior The effect of mobile phase pH and isoelectric point of the proteins are the parameters least studied in HIC. At the start of HIC method, it was reported that there is no obvious relationship between retention and isoelectric point of individual proteins (68). However, in the follow up studies some clear pH effects were observed, where basic proteins has shown longer retention when the mobile phase pH was close to their pI and acidic proteins exhibited less retention with basic pH values. In other words, the protein retention was increased at 108
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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
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Chapter 3 Table 5. List of the iden
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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 and 112: Chapter 3 3.3. Protein distribution
Page 113 and 114: Chapter 3 3.3.2.2. Protein distribu
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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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