Lipid Profiling Extraction Method for Animal Tissue
This procedure is generalized. Please contact Mary Roth (please include a subject line) to inquire.
A .pdf of this method, including shipping directions, can be found here.
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 at -20°C (freezer). When you are ready to prepare for shipping, evaporate completely under nitrogen gas or a speedvac, and redissolve in about 1.0 ml chloroform. Transfer to 2.0 ml clear glass vial with Teflon-lined screw cap (for example: clear glass; 2 mL; solid PTFE-lined cap; 1/2 dr., Fisher Scientific catalog #03-391-7A, Thermo Scientific No.: B7800-1). ALWAYS let us know before you send your samples: email@example.com
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.
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.