This procedure is generalized. Please contact us at lipid@ksu.edu (please include a subject line) to inquire.
Use glass tubes with Teflon-lined screw caps.
To 0.8 parts cells/tissue (homogenized; see below) in aqueous solution, add 1 part chloroform and 2 parts methanol. Shake well, add 1 part chloroform and 1 part water. Shake, centrifuge at low speed for 5-10 min. Remove the lower layer. Add 1 part chloroform, shake, centrifuge, remove the lower layer. Add 1 part chloroform, shake, centrifuge, remove the lower layer. Wash the combined lower layers once with a small volume 1 M KCl and once with a small volume of water.
Fill tubes with nitrogen, store in
freezer; and transport to the KLRC Analytical Laboratory on dry ice.
Alternatively, if you need to ship the samples, evaporate the
solvent, fill the tubes to nitrogen, and ship on dry ice. You may
wish to consider transferring the samples to 2 ml vials with teflon-lined
caps for shipping. Please contact Mary Roth at
mrroth@ksu.edu before shipping.
Comments:
If you have trouble with emulsions, you may have to add some KCl or other salt to the extraction. If you have polyunsaturated samples, we suggest including 0.01% BHT (butylated hydroxytoluene) in the chloroform. You may or may not need to grind your tissue. If you need to do this, if is preferable to do it either on sample frozen in liquid nitrogen (e.g. mortar and pestle) or on sample already in solvent (e.g. Dounce or Potter-Elvehejem homogenizer), as this reduces any possible enzymatically induced hydrolysis during the grinding or homogenizing.
Safety tip:
Wear safety glasses and protective clothing,
especially when operating a Potter-Elvehejem homogenizer with the sample in solvent,
because the solvent can accidentally splatter. To avoid this, start slowly. |
