Hello, I'm going to extract lipid from HepG2 cell by Folch method. Usually, for tissue, we put 100 mg of tissue into glass vial, then add 5 mL of chloroform:methanol:distilled water (2:2:1 v/v). So, could anyone let me know how much cell is usually needed for the Folch, and the additional prep method before putting the cell into the vial, please? Thank you a lot in advance.
Rahmani Youcef
The tissue is homogenized with chloroform/methanol (2/1) to a final volume 20 times the volume of the tissue sample (1 g in 20 ml of solvent mixture). After dispersion, the whole mixture is agitated during 15-20 min in an orbital shaker at room temperature. The homogenate is either filtrated (funnel with a folded filter paper) or centrifuged to recover the liquid phase. The solvent is washed with 0.2 volume (4 ml for 20 ml) of water or better 0.9% NaCl solution. After vortexing some seconds, the mixture is centrifuged at low speed (2000 rpm) to separate the two phases. Remove the upper phase by siphoning and kept it to analyze gangliosides or small organic polar molecules. If necessary (need of removing labelled molecules...), rinse the interface one or two times with methanol/water (1/1) without mixing the whole preparation. After centrifugation and siphoning of the upper phase, the lower chloroform phase containing lipids is evaporated under vacuum in a rotary evaporator or under a nitrogen stream if the volume is under 2-3 ml.
folch method
0 votes
0 thanks
- Badges
- Science topic
- Similar topics
- Biological Science
- Anatomy
- Tissue
More Jeong-Eun Choi's questions See All
Similar questions and discussions