The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology 
The Hebrew University of Jerusalem
Dr. Mario Lebendiker  Tel: 972-2-6586920 

Endotoxin Clearance with Triton X-114 - Selected protocols from the literature

Single step procedure enabling metal chelate affinity purification and endotoxin clearance

T. Zimmerman et al.  Simultaneous metal chelate affinity purification and endotoxins clearance of recombinant antibody fragments                                                                         Journal of Immunological Methods 314 (2006) 67–73 (pdf)

Wash IMAC (Ni) column with 50 column volumes of buffer containing 0.1% (v/v) of TritonX-114 (Sigma-Aldrich, Germany) followed by 20 column
volumes of buffer without detergent at 4 °C before Imidazole elution


Harvest of inclusion bodies, removal of endotoxin and solubilization

Z. Chen et al.  Efficient production of recombinant IL-21 proteins for pre-clinical studies by a two-step dilution refolding method.                                                                          Int Immunopharmacol (2013),  (pdf)

Cultured cells were pelleted by centrifugation (4000 g, 15 min) and resuspended in the lysis buffer (0.1 M Tris–HCl, pH 7.5, 1 mM EDTA) at 4 °C. Lysozyme
(1.5 mg/g of pellets) was added and incubated for 30 min. After sonication and incubation in the buffer A (0.5 M NaCl, 10 mM EDTA, 2% Triton X100)
for 30 min, inclusion bodies (IBs) were pelleted by centrifugation (30,000 g, 10 min) and resuspended in the buffer B (PBS pH 7.4, 1% Triton X-114).
Endotoxin was removed by repeating the following cycle for three times: incubation (4 °C, 30 min), heating (56 °C, 10 min), centrifugation (30,000 g, 10 min)
and collection of pellets [18]. Processed IB pellets were washed with PBS, resuspended in the solubilization buffer (PBS pH 7.4, 6 M guanidine hydrochloride
(Gdn-HCl)) and stirred at room temperature till solution came clear. After centrifugation (30,000 g, 10 min) to remove insoluble debris, the solution of
denatured proteins was filtered (0.45 μm) and loaded into Nickel-affinity chromatography (Qiagen, Valencia, CA, USA). The column was washed with
40 volumes of pre-chilled buffer C (PBS pH 5.3, 8 M urea, 0.1% triton X114) to further remove endotoxin [19]. After wash with 20 volumes of the buffer D
(PBS pH 5.3, 8 M urea) to remove the detergent, purified denatured IL-21 proteins were eluted by the buffer E (PBS pH 3.2, 8 M urea)

Phase separation technique to reduce endotoxin in protein solutions

Y. Aida and M. Pabst Removal of endotoxin from protein solutions by phase separation using Triton X-114                                                                                  
            Journal of lmmunological Methods, 132 (1990) 191-195 

Test solutions (0.5 ml) were mixed with Triton X-114 (Sigma) at 1% (5 gl) by vigorous vortexing in 1.5 ml conical microfuge tubes. Samples were placed in an ice
bath for 5 rain to ensure a homogenous solution. After vortexing the chilled samples, the tubes were warmed at 37°C for 5 min to allow two phases to form.
Samples were then centrifuged for 7 s with a microcentrifuge, equilibrated at 37°C in an incubator. In some experiments, tubes were placed in a 56°C water bath
for 1 min before centrifugation. After centrifugation, the detergent phase was found as an oily droplet at the bottom of the tube. The upper aqueous phase was
removed with care so as not to aspirate the detergent phase. Residual detergent in the aqueous phase was removed by gel filtration on BioGel P-300
(Bio-Rad Laboratories, Richmond, CA) or by treatment with Bio-Beads SM-4 (Bio-Rad)

At temperatures below 20°C, Triton X-114 detergentwill dissolve in aqueous solutions. At temperatures above 20°C, the detergent separates into two phases where

lipophilic endotoxin partitions to the organic phase.




Dr. Mario Lebendiker The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology,
The Hebrew University of Jerusalem  Tel: 972-2-6586920  

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