1 / 27

Lipid extraction at the ILL Progress report since Jan 2017

Lipid extraction at the ILL Progress report since Jan 2017. Rachel Morrison, Valérie Laux & Giovanna Fragneto Soft Matter Science and Support Group ILL, Grenoble. Lipid extraction. Freeze-dry & grinding. Cell pretreatment. Sonication. Glass beads & homogenization.

janeblack
Download Presentation

Lipid extraction at the ILL Progress report since Jan 2017

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Lipid extraction at the ILLProgress report since Jan 2017 Rachel Morrison, ValérieLaux & Giovanna Fragneto Soft Matter Science and Support Group ILL, Grenoble

  2. Lipid extraction Freeze-dry & grinding Cell pretreatment Sonication Glass beads & homogenization Courtesy Rachel Morrison

  3. Lipid extraction Freeze-dry & grinding Non-polar Cell pretreatment PG PE Sonication PS PC Lyso PC Glass beads & homogenization Beads + Blight Dyer Sonication + Blight Dyer Sonication + Folch Standards Courtesy Rachel Morrison

  4. Lipid extraction Freeze-dry & grinding Cell pretreatment Denaturation of lipases Sonication Glass beads & homogenization Courtesy Rachel Morrison

  5. Lipid extraction Non-polar Blight Dyer Extract PG PE Folch Extract PS PC Lyso PC Soxtec™ automated extraction Beads + Blight Dyer Sonication + Blight Dyer Sonication + Folch Standards Courtesy Rachel Morrison

  6. Lipid extraction Blight Dyer Extract Drying Separated and analyzed Total lipid extract Folch Extract Soxtec™ automated extraction Courtesy Rachel Morrison

  7. Lipid purification Image from Triebl et al. Biochimica et Biophysica Acta, 2017 Vol 1862 Courtesy Rachel Morrison

  8. Lipid class purification HPLC Solid Phase Extraction Courtesy Rachel Morrison

  9. Solid Phase Extraction • Aminopropyl AP (NH2) Column • Pre-conditioned in Hexane • Solvent 1- 30 ml Acetonitrile-IPA (2:1) • Solvent 2- 10 ml Methanol • Solvent 3- 7.5 ml IPA-Methanol 3N HCl 1 2 3 Courtesy Rachel Morrison

  10. HPLC- Silica column Operating conditions 0.5 ml/min until 60 mins 1.5 ml/min until 120 mins Solvents Hexane 367 Isopropanol 490 25mM phosphate 62 Ethanol 100 Acetic acid 0.6 Courtesy Rachel Morrison

  11. HPLC- Silica column Non-polar PE Peak 2 Peak 3 PC PS Peak 4 Lyso PC Peak 4 Peak 2 Peak 3 Courtesy Rachel Morrison

  12. HPLC- Diol column Operating conditions Diol column A: hexane – 2-propanol – butanol – THF – iso-octane – water (64.5:17.5:7:5:5:1) B: 2-propanol – butanol – THF – iso-octane – water (73:7:5:5:10)ammonium acetate was added to both solvents (180 mg/L)Temperature: 65 °C Courtesy Rachel Morrison

  13. HPLC- Diol column Courtesy Rachel Morrison

  14. HPLC- Diol column PC Non-polar PS PE PE PS PC Peak 4 Peak 3 Peak 5

  15. HPLC- Silica column • Currently evaluating Si column • Acetonitrile, Methanol, phosphoric acid Courtesy Rachel Morrison

  16. Lipid characterization • Thin layer chromatography, • Gas chromatography, • Ellipsometry, • IR spectroscopy, • Mass spectroscopy, • Light scattering, • Neutron and x-ray scattering. Courtesy Rachel Morrison

  17. GC acquired from Shimatsu, to be installed on 7th March in PSCM labs – training 8th March Courtesy Rachel Morrison

  18. Large scale production of deuterated lipids for use by the user community Si SiO2 Neutron experiments Fermenters for large batches of cell paste H2O Reconstruction of model membranes Courtesy Rachel Morrison

  19. Future work- Fatty acid incorporation into phospholipids • Yeast grown with • H-Palmitic acid (C16:0) • D-Palmitic acid (C16:0) • H-Oleic acid (C18:1) Courtesy Rachel Morrison

  20. Future work- Fatty acid incorporation into phospholipids • Yeast grown with • H-Palmitic acid (C16:0) • D-Palmitic acid (C16:0) • H-Oleic acid (C18:1) Reproduced from data in Leitner et al. Biochimica et Biophysica Acta, 2007, Vol 1771 Courtesy Rachel Morrison

  21. Future work- Fatty acid incorporation into phospholipids • Yeast grown with • H-Palmitic acid • D-Palmitic acid • H-Oleic acid Courtesy Rachel Morrison

  22. 18 January 2017 Summary and future work • Cell cultures grown in perdeuterated media • Optimisation of lipid extraction protocols • Developing purification methods for lipids • Investigate the effect of fatty acid feed on resulting phospholipid production. Courtesy Rachel Morrison

  23. Characterisation of extracts by neutron diffraction on D16

  24. Kiesel et al., submitted

  25. Rachel Morrison on leave from 08/17 to 02/18 (contract extended until end of 04/18) New post-doc hired: Krishna Batchu • To arrive 1st June 2018 • PhD in LipidBiochemistry (Helsinki Institute of Biomedicine) • MSc of Engineering in Bioinformatics (Chalmers) • 10 yearsexperience in LipidBiochemistry/Lipidomics • Experience HPLC, TLC, Mass Spec, Solid Phase Extra(D9) PC species, …

  26. Next ILL deliverable 5.9 :Optimization of separation and purification methodsdue in Sept 2018 (month 36)Can we move it to month 42 (to recover the months of leave of post-doc)?

  27. Aknowledgements PSCM at the ILL Rachel Morrison SINE2020 post-doc Michael Haertlein D-Lab Platform Head D-Lab at the ILL Yuri Gerelli PSCM Coordinator David Hess Laboratory Responsible Valerie Laux D-Lab Research Technician Robin Delhom Former PhD Student

More Related