A variety of methods have been developed in order to remove endotoxins from protein solutions, with one of the more popular being separation by phase using non-ionic detergent Triton X-114. Triton X-114 detergent is homogeneous at cold temperatures, but once above a critical temperature of 22°C, its solution separates into an aqueous phase, where hydrophilic proteins are found, and a detergent phase, where impurities such as endotoxins and other hydrophobic molecules are captured. Multiple cycles of cooling-heating-phase separation have to be repeated until the remaining endotoxin is below the threshold limit. It should be noted that this method cannot be used if a target protein happens to be hydrophobic and partitions into a detergent-enriched phase.
This is a simple and cheap procedure. In most cases the repeated cooling heating process has proven to remove endotoxins without affecting any significant losses in protein yields and biological activities. However, it has to be noted that residual Triton will remain in the sample at <0.02% concentration and it cannot be dialyzed out due to its large micelle size.
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