Biochemistry

scientificprotocols authored over 3 years ago

Authors: Wei Zou

Abstract

SPE is an important technique to separate different classes of lipids after total lipid extraction.

Reagents

  1. Chloroform: methanol W/BHT 2:1 = 2 L: 1 L:100 mg
  2. Acetone: water (ACW) = 7:1 as 700 mL:100 mL (v/v)
  3. Hexane
  4. Hexane:methyl tert-butyl ether:acetic acid (HBA) = 100:3:0.3 (v/v/v) as 1000 mL hexane:30 mL methyl butylether:3 mL acetic acid
  5. Hexane: chloroform:ethyl acetate (HCA) = 100:5:5 (v/v/v) as 800 mL hexane:40 mL chloroform:40 mL ethyl acetate
  6. Chloroform:2-propanol (CIP) = 2:1 (v/v/v) as 600 mL chloroform:300 mL 2-propanol
  7. Chloroform:methanol:acetic acid (CMA) = 100:2:2 (v/v/v) as 750 mL chloroform:15 mL methanol:15 mL acetic acid
  8. Methanol:chloroform:water (MCW) = 10:5:4 (v/v/v) as 500 mL methanol:250 mL chloroform:200 mL water
  9. Bond Elut NH2-Aminopropyl column (Varian Sample Preparation Products, Harbor City, CA USA)
    • 1g capacity columns are for liver samples, while 500 mg capacity columns are for bile samples.
  10. Test tube with Teflon cap (16 mm x 100 mm)
  11. Vac Elut SPS 24TM Vacuum elution apparatus (Varian Sample Preparation Products, Harbor City, CA USA)

Procedure

  1. Determine the total lipid concentration in liver lipid extraction or bile.
    • SPE method allows lipid concentration <1.46 mg/100µl.
  2. Prepare new solvent in dispenser from stock solution.
    • Throw away the old solvent because it is easy to evaporate in dispenser than in stock bottle.
  3. Take proper amount of liver lipid extraction or bile. Evaporate under N2. Add 900µl HBA into the tube.
  4. Connect the SPE vacuum apparatus to the negative water pressure system. Switch on the pressure control. (Don’t exceed 2)
  5. Switch the upper part of SPE vacuum apparatus to “Waste” position.
  6. Use 1000 µL automatic pipette to add 600 µL ACW twice to wash the SPE column.
  7. Change the switch on the SPE vacuum apparatus from WASTE to COLLECTIVE.
  8. Use 1000 µL automatic pipette to add 1000 µL hexane twice.
  9. Apply 300 µL lipid HBA (LHBA) into the column.
  10. Set up the tubes of CE fraction into the SPE vacuum apparatus.
  11. Use dispenser to add 5 mL hexane into the column to elute CE. Wait until all the solution is eluted.
  12. Change the tubes to TG. Use dispenser to add 5 mL HCA.
  13. Change the tubes to MDG. Use dispenser to add 5 mL CIP.
  14. Change the tubes to FFA. Use dispenser to add 5 mL CMA.
  15. Change the tubes to PL. Use dispenser to add 5 mL MCW.
  16. For PL fraction, add 2 mL water + 1 mL chloroform to separate water in the PL fraction. Shake upside down 3 times. Siphon off the upper layer. Cap tightly and seal it with Teflon tape. Evaporate the lower layer for FA analysis.

References

  1. Zou, W., Noh, S.K., Owen, K.O., & Koo, S.I. (2005) Dietary carnitine enhances the lymphatic absorption of fat and a-tocopherol in ovariectomized rats. J. Nutr. 135: 753-756. http://jn.nutrition.org/content/135/4/753.full
  2. Folch, J., Lees, M., and Sloane-Stanley, G.H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509.
  3. Agren, J.J., Julkunen, A., and Penttila, I. (1992) Rapid separation of serum lipids for fatty acid analysis by a single aminopropyl column. J. Lipid Res. 33:1871-1876.

Acknowledgements

Thanks to Koo, S.I. for mentoring and financial supporting efforts.

Author information

Wei Zou, Zou's lab, DTSC, California State Government

Correspondence to: Wei Zou ([email protected])

Source: Protocol Exchange (2011) doi:10.1038/protex.2011.246. Originally published online 28 July 2011.

Average rating 0 ratings