Lipid Analysis of Manduca Sexta Midgut

Procedures: Day 1

Friday, April 26, 2002 8:00-4:30

  1. Prepare Thin Layer Chromatography (TLC) chamber with petroleum ether:ethyl ether:acetic acid (80 mL : 20 mL : 1 mL).
  2. Dissect Manduca sexta (tobacco hornworm), remove midgut and rinse with potassium phosphate buffer (K2HPO4), pH =8.0.
  3. Mass midgut tissue:  0.1423 g
  4. Suspend in 3 mL chloroform (CHCl3)/methanol (MetOH) 2:1 (v/v)  
  5. Homogenize using a tissue grinder
  6. Sonicate (pulse: 0.5 sec / output: 20 watts / 55 sec. duration)
  7. Add 1 mL nanopure water
  8. Vortex for 15 seconds & centrifuge for 5 min. @ 1300 xg
  9. Extract bottom layer with a pasteur pipet (chloroform/lipid)
  10. Add 2 mL CHCl3, sonicate, centrifuge, extract bottom layer (x2)
  11. Evaporate CHCl3 solvent under N2 using an N-Evap apparatus
  12. Prepare TLC Plates (three lanes: lipid standard 1, sample, lipid standard 2)
  13. Add 100 mL CHCl3, vortex, centrifuge, then spot TLC plate
  14. Add 75 mL CHCl3, vortexed, centrifuged, respotted plate (x2)
  15. Place in TLC chamber for about an hour (until solvent front moves to top of lanes)
  16. Remove plate and allow it to dry
  17. Start a dry bath @ 92 degrees Celsius
  18. Place plate in an iodine chamber to expose unsaturated lipids
  19. Label bands according to standards composition (monoglycerides (monolein), diglycerides (diolein), cholesterol, free fatty acid (oleic acid), triglyceride (triolein), methyl oleate, cholesterol oleate)
  20. Scrape phospholipids (on/about the origin) and diglycerides from the plate into vials
  21. Add 3 mL MetOH and 3 drops of H2SO4 to each vial
  22. Seal vials under N2 (prevents oxidation by O2)
  23. Reflux @ 91 degrees Celsius for 2 hours
  24. Remove vials, cool, add 1 mL H20 and 2 mL petroleum ether
  25. Vortex, centrifuge @ 1300 xg for 2 minutes, and collect top fraction (x3)
  26. Stored in freezer overnight @ -20 degrees Celsius