Mechanisms of Olfaction in Insects - ResearchSpace@Auckland ...

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The roles of Epiphyas postvittana GOBP2 in odour detection 66 Figure 3.2: Purification of His6-EpGOBP2 by HiTrap chelating HP column. A dominant EpGOBP2 band is observed at 22.6 kDa in lanes 4–9. Lane 1 is the insoluble fraction containing the cell debris, lane 2 is the crude protein sample that was loaded onto the column and lane 3 is the flow through. His6-tagged EpGOBP2 was present in all six fractions over the elution peak, shown by a dominant band at 22.6 kDa. However, other bands were present in the fractions also, possibly E.coli proteins that interacted with the nickel column. To further purify EpGOBP2, the six fractions from the HiTrap chelating column were combined and dialysed into ion exchange binding buffer for purification on Q-sepharose HP column (Amersham Biosciences). The protein was eluted using a gradient of 500 mM NaCl. The seven elution peak fractions obtained were run on 4–12% a SDS-PAGE gel as shown in Figure 3.3.
The roles of Epiphyas postvittana GOBP2 in odour detection 67 Figure 3.3: Purification of His6-EpGOBP2 from the pooled HiTrap fractions by Qsepharose anion exchange chromatography. Lane 1 is the EpGOBP2 dialysed into ion exchange binding buffer and lanes 2–8 are the elution peak fractions from the Qsepharose HP column containing the His6-EpGOBP2. The fractions obtained after the ion exchange were pooled and the His6-tag was cleaved off by AcTEV digestion. After tag removal, the protein was recovered by running the sample on HiTrap chelating HP column, this time collecting the protein in the flow through (Figure 3.4I). The protein sample was finally put through a delipidation procedure to remove any exogenous ligands that bound to the protein from the E. coli cells (Figure 3.4II).
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Abstract Abstract Olfaction or the
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Acknowledgements Acknowledgements I
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Contents v Contents Abstract……
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Contents 4.2.5 Degenerate PCR .....
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List of Figures List of Figures Fig
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List of Tables List of Tables Table
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List of Abbreviations gDNA Genomic
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1.1 General overview 1 General Intr
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General Introduction 3 moth in loca
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General Introduction 5 Figure 1.1:
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General Introduction 7 1.5 Componen
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General Introduction 9 OBPs have al
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General Introduction 11 A model has
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General Introduction 13 Figure 1.3:
Page 31 and 32: General Introduction 15 Tanaka et a
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Page 37 and 38: General Introduction 21 et al., 200
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Page 45 and 46: General Introduction 29 Expression
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Page 51 and 52: Functional characterisation of Epip
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Identification of putative odorant
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5.1 Introduction 5 Concluding Discu
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Concluding Discussion 127 VOBA sett
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Concluding Discussion 129 redundant
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Concluding Discussion 131 against t
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Concluding Discussion 133 sequencin
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Appendix B 135 Buffered TB (Sambroo
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Appendix C 137 C. Appendix C - Clus
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Appendix D 139 D. Appendix D - Solu
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Appendix E 141 I II
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Appendix E 143 V
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Appendix E 145 VII continued Figure
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References 147 Ansebo, L., Ignell,
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References 149 Butenandt, A., Beckm
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References 151 Fedurco, M., Romieu,
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References 153 Große-Wilde, E., St
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References 155 Hrdy, I., F. Kuldova
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References 157 Kaissling, K. E. (19
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References 159 Krieger, J., Große-
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References 161 Lopez-Gomez, R. and
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References 163 Miura, N., Nakagawa,
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References 165 Pell, J. K., Macaula
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References 167 Røstelien, T., Stra
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References 169 Stowers, L. and Loga
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References 171 Turner, C. T., Davy,
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References 173 Vogt, R. G., Riddifo
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References 175 Widmayer, P., Heifet
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