Suggested extraction procedures For research use only. Not for use in diagnostic procedures.
Extraction Protocol for Plasma Using SPLASH ™ or UltimateSPLASH™ ONE 1. Use 13 x 100 mm new glass screw capped tubes. Do not use washed tubes as you may extract detergent residue. 2. Add 990 μl water to 10 μl plasma, then let sit on ice for 10 minutes. 3. Add 2.0 mL methanol. 4. Add 0.9 mL dichloromethane. 5. Vortex. 6. A singe phase should appear. If there are two distinct phases, add 50 μl methanol and vortex. If solution is still not a single phase, repeat addition of 50 μl methanol and vortex. 7. Add 10 μl UltimateSPLASH™ ONE Internal Standard, vortex, and let mixture sit for 30 minutes at room temperature. 8. Add 1 mL water. 9. Add 0.9 mL dichloromethane.
4. For cells in suspension: centrifuge, discard saline, and add 1 mL water. Vortex and transfer to glass tube for extraction. Rest on ice for 10 minutes. Ensure final volume is 1 mL and adjust if necessary. 5. For adhered cells: wash cells with non-buffered saline. Add 1 mL water to lyse cells and scrap. Collect cell lysate and transfer to glass tube for extraction. Rest on ice for 10 minutes. Ensure final volume is 1 mL and adjust if necessary. 6. Add 2.0 mL methanol. Add 990 μl water to 10 μl plasma, then let sit on ice for 10 minutes. 7. Add 0.9 mL of dichloromethane. 8. Vortex. 9. Repeat steps 6-16 from Extraction Protocol for Plasma. Extraction Protocol for Cell Culture 1. Weigh tissue to be extracted. 50-100 mg is sufficient. Calculate water content. Expected values are as follows: a. Adipose 18% b. Brain 60% c. Bone 44% d. Heart, kidney, liver, lung, intestines, spleen, and stomach 65% e. Testes 18% 2. Add water to tissue so that total volume is 1 mL. Example: 100 mg of brain tissue corresponds to 60 μL water. Add 940 μL water. 3. Disperse tissue. a. Grind tissue frozen in liquid nitrogen using cold mortar and pestle. b. Blend using a homogenizer. 4. Sonicate for 30 seconds with 5 seconds bursts and 20 second rest time. Perform sonication steps on ice. 5. Add 2.0 mL of methanol. 6. Add 0.9 mL dichloromethane. 7. Vortex. 8. Repeat steps 6-16 from Extraction Protocol for Plasma.
10.Invert tube 10 times. DO NOT VORTEX. 11.Centrifuge at 1200 rpm for 10 minutes.
12.Collect lower layer and put into a new glass tube. 13.Add 2 mL dichloromethane to remains in extraction tube. 14.Mix, centrifuge, and collect lower layer. Add to first extract. 15.Evaporate solvent under a stream of nitrogen. 16.Re-suspend lipids in injection solvent. Extraction Protocol for Cell Culture 1. Use 13 x 100 mm new glass screw capped tubes. Do not use washed tubes as you may extract detergent residue. 2. Collect cells. 3. Wash cells with non-buffered saline to remove cell culture
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