I want to extract the the degraded plasma membrane of K562 cell lines without any of the cell organelles or extracellular constituents as I want to use the membrane for lipidomic analysis.
Sucrose step gradient ultracentrifugation is a good way to purify plasma membrane away from other membranes. When I used to do this with CHO cells, the plasma membrane was the lightest fraction and was isolated from the 16%/31% sucrose interface.
Adam B Shapiro thank you. Could you please tell me how to prepare the gradient manually? What type of tubes will I have to use?
Ultraclear (Beckman) tubes should be used so that you can see through them from the side. You will need a swinging bucket rotor for the ultracentrifuge, and the size of the tubes should be appropriate for the rotor. Start by adding the highest sucrose solution to each tube. Layer the next highest concentration sucrose solution on top of that, very gently to avoid mixing the two solutions together. Use a Pasteur pipette with a rubber bulb, or a disposable transfer pipette in order to have control over the liquid dispensing. Continue adding each sucrose step. Leave room at the top for the sample. The tubes should be almost completely filled. All the tubes should have the same volume and weight.
Concerning the use of swinging bucket ultracentrifuge rotors: If you have not used one before, seek guidance from an experienced person to avoid disaster. Always put all the buckets on the rotor, even if some of them are empty. Loading the rotor onto the centrifuge spindle without disturbing the tubes can be tricky. Practice it before doing it with your precious samples.
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