In most of the commercial kits, they have a proprietary formulation which is not disclosed.
The removal of endotoxin by means of specific columns is based on immobilized polymyxin B, which has a high affinity for endotoxin.The columns can usually be regenerated with deoxycholate, as specified in the manufacturer's instructions. However, the binding capacity of such columns is sometimes to small to remove large amounts of endotoxins present in some preparations of recombinant proteins obtained from recombinant E. coli. In such cases, it is advisable to proceed with a wash of the immobilized recombinant protein with buffer containing 0.1 % Triton X-114, followed by a wash with Triton-free buffer until the A280 due to the presence of Triton X-114 nearly disappears (A280 < 0.03). The remaining traces of endotoxin may the be removed using a polymyxin-agarose column.
Pierre Béguin Thank you sir. May I ask this out of curiosity, is this the method (0.1% Triton wash) used by pharma industry to prepare endotoxin free proteins for human use, or that is different?
To Joshua Paul: I don't know what techniques are used by the industry. there are papers describing the use of Triton X-114 for extraction of endotoxin by phase partitioning. I tried phase partitioning but found that in my hands, on-column washing gave me better resuilts in terms of yield and endotoxin elimination
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