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

More documents

Recommendations

Info

The roles of Epiphyas postvittana GOBP2 in odour detection 62 3.2.1.1 Purification of recombinant EpGOBP2 The cell pellet was resuspended in 20 mL HisTrap binding buffer (consisting of 20 mM Tris pH 8.0, 500 mM NaCl and 20 mM imidazole) containing EDTA free protease inhibitors (Roche). This solution was emulsified at 15000 psi by passing the samples through an emulsiflex-CS high-pressure homogeniser (Avestic Inc.) three times to get maximum cell lysis. The sample was then centrifuged at 13,000g at 4°C for 30 minutes, the resulting pellet discarded and the soluble fraction filtered through a 0.22 µm filter (Millipore) to remove any remaining cell debris and kept on ice until purification. Purification of the His6-EpGOBP2 was carried out on a 5 mL HiTrap chelating HP column (GE Healthcare) by affinity chromatography on ÄKTAprime fast protein liquid chromatography (GE Healthcare). The protein solution was loaded onto the column equilibrated in HisTrap binding buffer containing 20 mM imidazole. Bound His6-EpGOBP2 was eluted from the column by a 20 to 250 mM imidazole linear elution gradient. The fractions containing His6-EpGOBP2 were identified by running on 4-12% Bis-Tris SDS PAGE gel (Invitrogen) and visualised using colloidal commassie G-250 blue stain (Neuhoff et al., 1988). Fractions showing the presence of His6-EpGOBP2 were pooled and dialysed overnight into ion exchange binding buffer consisting of 20mM Tris HCl, pH 8 and 25 mM NaCl and loaded onto a Q sepharose HP column (GE Healthcare). Bound protein was eluted from the column by a 25 to 500 mM NaCl linear elution gradient. The fractions containing His6-EpGOBP2 were identified by SDS-PAGE analysis, pooled and then dialysed against 50 mM Tris-HCl pH 8.0, 0.5 mM EDTA overnight. After dialysis, the His6-tag was cleaved off with AcTEV Protease (Invitrogen) following manufacturer‟s instructions. After cleavage, the protein sample was dialysed into HisTrap binding buffer for purification of the tag free protein on the HiTrap chelating HP column as stated above, this time collecting the protein in the flow through. 3.2.1.2 Delipidation of recombinant EpGOBP2 Recombinant OBPs have a tendency to bind exogenous ligands from the expression cells hence these artefact ligands have to be removed from the proteins before any
The roles of Epiphyas postvittana GOBP2 in odour detection 63 functional analysis is carried out (Oldham et al., 2000; Hamiaux et al., 2009). The tag-cleaved protein was delipidated according to Oldham et al. (2001). Briefly, the protein was concentrated to 50 µL using a Vivaspin ultrafiltration concentrator (capacity 500 µL, MWCO 5 kDa) at 10,000 rpm, 4°C (Vivaproducts, MA). 500 µL of 25 mM ammonium acetate pH 4.5 was then added to the retentate and centrifuged again to 50 µL. This was repeated a second time and then 350 µL of 25 mM ammonium acetate pH 4.5 was added again to the retentate and placed on ice. 500 µL of the Lipidex 1000 methanol suspension (Perkin Elmer) was centrifuged in a MC- Ultrafree centrifuge filter cup (capacity 500 µL, pore size 0.22 µm, Millipore, Bedford, MA) at 10,000 rpm for one minute to remove the liquid phase. The flow through was discarded and another 500 µL of Lipidex was added to the filter cup, centrifuged and the flow through discarded again. The Lipidex in the filter cup was mixed with 500 µL of 25 mM ammonium acetate pH 4.5, centrifuged at 10,000 rpm, for one minute and the flow through discarded. This was repeated again and then the EpGOBP2 sample was mixed thoroughly with the Lipidex, and the resulting suspension incubated in the filter cup at 37°C for one hour with gentle shaking. The suspension was then centrifuged at 10,000 rpm, 4°C; the flow through collected and kept on ice. The Lipidex was washed with 500 µL of 25 mM ammonium acetate pH 4.5 (to remove any remaining traces of EpGOBP2), centrifuged and flow through collected again. Both the flow through samples were combined and concentrated using the Vivaspin ultrafiltration concentrator to 50 µL. The concentrated, delipidated EpGOBP2 was diluted in 2.5 mM ammonium acetate at neutral pH to a concentration of 100 µM and dialysed against 50 mM Tris-HCl buffer pH 7.5 for conducting binding assays. 3.2.2 Volatile odorant binding assay The volatile odorant binding assay (VOBA) (Briand et al., 2000) was used for testing the ligand preferences of EpGOBP2. Fifty microlitres of 5 µM protein in 50 mM Tris- HCl, pH 7.5 or 50 µL of 50 mM Tris-HCl buffer pH 7.5 (control) were setup in triplicates in 200 µL PCR tubes. The tubes were placed with lids open in Conway microdiffusion dish (Gallenkamp). A drop of the absolute compound to be tested was added to the dish and the system was sealed with a glass lid. The system was left to equilibrate for approximately 12 hours at room temperature, as depicted in Figure 3.1.
Page 1 and 2:
http://researchspace.auckland.ac.nz
Page 3 and 4:
Abstract Abstract Olfaction or the
Page 5 and 6:
Acknowledgements Acknowledgements I
Page 7 and 8:
Contents v Contents Abstract……
Page 9 and 10:
Contents 4.2.5 Degenerate PCR .....
Page 11 and 12:
List of Figures List of Figures Fig
Page 13 and 14:
List of Tables List of Tables Table
Page 15 and 16:
List of Abbreviations gDNA Genomic
Page 17 and 18:
1.1 General overview 1 General Intr
Page 19 and 20:
General Introduction 3 moth in loca
Page 21 and 22:
General Introduction 5 Figure 1.1:
Page 23 and 24:
General Introduction 7 1.5 Componen
Page 25 and 26:
General Introduction 9 OBPs have al
Page 27 and 28: General Introduction 11 A model has
Page 29 and 30: General Introduction 13 Figure 1.3:
Page 31 and 32: General Introduction 15 Tanaka et a
Page 33 and 34: General Introduction 17 Table 1.1:
Page 37 and 38: General Introduction 21 et al., 200
Page 39 and 40: General Introduction 23 the attract
Page 41 and 42: General Introduction 25 leaves and
Page 45 and 46: General Introduction 29 Expression
Page 47 and 48: General Introduction 31 BmOr2 HvOr2
Page 49 and 50: General Introduction 33 for lepidop
Page 51 and 52: Functional characterisation of Epip
Page 73 and 74: 3.1 Introduction 3 The roles of Epi
Page 75 and 76: The roles of Epiphyas postvittana G
Page 77: The roles of Epiphyas postvittana G
Page 95 and 96: 4.1 Introduction 4 Identification o
Page 97 and 98: Identification of putative odorant
Page 109 and 110: Microarray OR 454 Transcriptome OR
Page 129 and 130:
Identification of putative odorant
Page 131 and 132:
Page 133 and 134:
Page 135 and 136:
Page 137 and 138:
Page 139 and 140:
Page 141 and 142:
5.1 Introduction 5 Concluding Discu
Page 143 and 144:
Concluding Discussion 127 VOBA sett
Page 145 and 146:
Concluding Discussion 129 redundant
Page 147 and 148:
Concluding Discussion 131 against t
Page 149 and 150:
Concluding Discussion 133 sequencin
Page 151 and 152:
Appendix B 135 Buffered TB (Sambroo
Page 153 and 154:
Appendix C 137 C. Appendix C - Clus
Page 155 and 156:
Appendix D 139 D. Appendix D - Solu
Page 157 and 158:
Appendix E 141 I II
Page 159 and 160:
Appendix E 143 V
Page 161 and 162:
Appendix E 145 VII continued Figure
Page 163 and 164:
References 147 Ansebo, L., Ignell,
Page 165 and 166:
References 149 Butenandt, A., Beckm
Page 167 and 168:
References 151 Fedurco, M., Romieu,
Page 169 and 170:
References 153 Große-Wilde, E., St
Page 171 and 172:
References 155 Hrdy, I., F. Kuldova
Page 173 and 174:
References 157 Kaissling, K. E. (19
Page 175 and 176:
References 159 Krieger, J., Große-
Page 177 and 178:
References 161 Lopez-Gomez, R. and
Page 179 and 180:
References 163 Miura, N., Nakagawa,
Page 181 and 182:
References 165 Pell, J. K., Macaula
Page 183 and 184:
References 167 Røstelien, T., Stra
Page 185 and 186:
References 169 Stowers, L. and Loga
Page 187 and 188:
References 171 Turner, C. T., Davy,
Page 189 and 190:
References 173 Vogt, R. G., Riddifo
Page 191 and 192:
References 175 Widmayer, P., Heifet
show all

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

Create successful ePaper yourself

Delete template?

Save as template?