Triton X-114 Partitioning of Farnesylated Proteins

Triton X-114 equilibrated with PBS is diluted to a 20 % stock solution (Madshus et al., 1984). 

To separate the phases, the solution is incubated tor 15 min at 37 °C, and then centrituged tor 2 min at room temperature in an Eppendort centrituge. 

The upper (water) phase is transterred to a new tube, and the lower (Triton) phase is washed at 37 °C with 500. 1 PBS. 

Both phases are then diluted to 1 ml with PBS (0 C), followed by immunoprecipitation with antibodies adsorbed to protein A-Sepharose. The precipitated material is analyzed with SDS-PAGE and tiuorography. 

To study partitioning ot proteins trom cells, the cells are lysed directly in the Triton/PBS mixture in the presence ot 1 mM PMSF, and, after removal of the nuclei by centrifugation, the procedure tor phase separation described above is followed. It should be noted that many antibodies do not bind well to their antigens in the presence of high concentrations ot detergent. It is therefore advisable to dilute the Triton X-114 phase with water at 0 °C before immunoabsorbtion. 

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