We are trying to create a method to separate high and low lipid-producing yeast cells after EMS treatment (mutagenesis). We have seen cells that accumulate up to 70% of their dry-weight in lipid bodies alone. Therefore, we suspect that cells that produce a high quantity of lipids have a higher buoyancy and therefore remain on the surface of a liquid medium (following centrifugation), while the low lipid-producing cells remain in the pellet.
We have tried using water as a 'medium' to facilitate the difference in buoyancy, but here we could not find cells on the surface. We have also tried different concentrations of sucrose and glycerol (which are denser), but this sadly rendered the same result.
My question is: Is there a significant difference in buoyancy between cells with a high/low quantity of lipid bodies? And if so, is there a suitable method to separate high-producers from low-producers?
Thank you in advance!
I think isopycnic Percoll ultracentrifugation gradients would allow you to answer these questions. Make sure to disrupt your cells into one-cell population. Load the corresponding number of individual cells in an HBS-Percoll gradient (Hepes buffered saline-Percoll). Cover and put in ultracentrifuge. Spin at almost maximal speed and isolate density bands and characterize their cells.
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