I tried to use triton X-114 phase separation method to remove LPS from my purified fusion protein. I followed the protocol as mentioned by Liu et al. In summary: Triton X-114 was added to the protein preparation to a final concentration of 1%. The mixture was incubated at 4° C for 30 min with constant stirring to ensure a homogeneous solution. The sample was then transferred to a 37° C water bath, incubated for 10 min, and centrifuged (20,000 g, 10 min) at 25° C. But after centrifuging no phase separation happened! I tried to change the triton source but nothing changed. Since my method of purification was denaturing, I'd like to know if urea presence in the protein buffer may interfere with the triton separation.