Phase Partitioning of Cells
Categorized: Outer Membranes
For assessing hydrophobicity of the cell surface.
- Grow cells to midlog phase, centrifuge and concentrate 10 fold.
- Prepare phases by adding volumes of 20% (w/w) polyethylene glycol 6000 Sigma) and 20% (w/w) Dextran T500 (Pharmacia) to give final concentrations of 4.4% and 6.2%, respectively, in 30mM Tris-HCl buffer, pH 7.0. Equilibriate at 4C. for several hours to allow separation into the 2 phases.
- Add 1 ml of each of the top and bottom phases. Add 0.1 ml of Triton X-100 and 0.1 ml of the concentrated cells.
- Invert tubes 20x and leave to equilibrate for 45 min. at room temperature.
Take a sample from both the top and the bottom phases. The OD of each of these samples is measured in a spectrophotometer at 600 nm. Results may be expressed as the ratio of the top/bottom phases.