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

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The roles of Epiphyas postvittana GOBP2 in odour detection 64 Figure 3.1: Schematic representation of the VOBA setting to show the distribution of the odorant at equilibrium in a sealed chamber. The tubes were removed after the 12 hour incubation. To release the bound compounds, 5 µL of 1 mg/mL proteinase K (Roche) was added to the tube content, which was then covered with a layer of hexane (to capture odorants released from digested protein). This was done for all the tubes, including the buffer only control. 5 µM of an internal standard, methyl tetradecanoate (14OMe) was added to all the tubes. The closed tubes were then incubated at room temperature for two hours. The hexane layer was removed from tubes into clean 2 mL glass vials and analyzed by GC using a HP 6890 Series GC System (Hewlett Packard) equipped with an on-column injector and FID detector (300°C). The analytical column used was a HP-INNOWax Polyethylene Glycol (nominal length = 30.0 m, nominal diameter = 250.00 µm and nominal film thickness = 0.25 µm; Agilent Technologies). The oven temperature gradient was applied from 120°C to 260°C at 5°C.min -1 . The carrier gas was helium at 0.7mL.min -1 . Calibration was done by odorants diluted in chloroform. The concentration of the compounds was determined by measuring surface of peaks with respect to the calibration curve. The concentration of the bound ligand and the binding affinity (dissociation constant, ) of the ligands with the protein were calculated using the method of Lazar (2001) as follows: Protein Test ligand Buffer
The roles of Epiphyas postvittana GOBP2 in odour detection 65 From the results obtained in the GC, [L] = free ligand (total ligand present in buffer only) [P] = protein concentration used for VOBA (5 µM) [PL] = concentration of bound ligand (Total ligand present in protein sample – [L]) = dissociation constant (the smaller the , the tighter the binding) 3.2.3 Reconstituted cell assay The reconstituted cell assay was carried out as described in Groβe–Wilde et al. (2006) with a few modifications. Briefly, odorants were made up in the concentration range of 10 -5 M to 10 -14 M as described in section 2.2.6 in either assay saline alone or assay saline containing 10 -6 M EpGOBP2 solution. The Sf9 cell transfection, assay and data analysis were performed as stated in sections 2.2.6 and 2.2.7. 3.3 Results 3.3.1 Purification of recombinant GOBP2 Recombinant EpGOBP2 was purified in order to carry out functional ligand binding assays to deduce its ligands and also to test its role(s) in odorant binding of EpOR1 in the Sf9 cell assay system. His6-EpGOBP2 eluted as a single peak from 60 to 75% imidazole. The six fractions collected during this elution peak were run on 4-12% SDS-PAGE gel (Figure 3.2). No EpGOBP2 was present in the flow through indicating all the EpGOBP2 loaded onto the column was bound to it. However, not all of the EpGOBP2 separated into the soluble phase as there is still a thick band in the insoluble fraction at 22.6 kDa.
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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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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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