Sayani Haldar , hello, your current problem is that after HepG2 cells were lysed with RIPA buffer containing NP-40, three layers (upper lipid layer, middle transparent protein layer, bottom debris layer) were formed, and lipids were mixed in when the middle layer was collected.

Basic suggestions:

Carefully aspirate the middle layer, use a slender pipette with a scale to stick close to the interface between the lipid and the transparent layer, and aspirate slowly. Do not touch the upper lipid layer. You can tilt the tube wall, concentrate the lipids on one side, and then aspirate from the other side.

If the middle layer still contains a small amount of lipids after aspiration, you can centrifuge it again at a higher speed (such as 12,000–14,000xg, 4°C, 10 minutes) (you can centrifuge it at a low speed multiple times) to make the lipids float and the bottom debris sink. Only take the clearer part in the middle.

Operation at 4°C throughout the process can solidify the lipids, make the interface clearer, and reduce contamination.

Optimization suggestions

Acetone precipitation delipidation method

After collecting the middle protein layer, add 4-6 times the volume of pre-cooled acetone, mix well and precipitate at -20°C for 1-2 hours, then centrifuge to collect the protein precipitate. This step can effectively wash away the mixed lipid impurities and most small molecules.

Density gradient centrifugation or washing method

When encountering samples with high fat content, you can try to cover the upper layer of the lysate with 1-2ml buffer and centrifuge slowly to make the lipid distribution more concentrated and easier to sample.

The answer to this question comes from MedChemExpress (MCE) Technical Support.

[email protected]