Hi all,
I want to remove the endotoxins from my protein sample, since they could overestimate the immune response of the antigen. I measured the endotoxin level in my sample after gel filtration, but is still positive to endotoxin content, which is very strange. Maybe the LPS is attaching to the protein.
Anyway, I have ordered an Endotrap Endotoxin Removal Kit, but I am wondering if exist another method for endotoxin removal that I could try in the meantime.
Many thanks,
Julen
Hello Julen,
Endotoxin removal from recombinant proteins produced in a recombinant E. coli host is indeed a common problem. My experience is that there can be so much of it that it will saturate the immobilized polymixin-based resin provided with the endotoxin removal kit. As a first step, it is therefore advisable to bind your protein to some resin like Ni-NTA and perform a wash with Triton X-114 followed by Triton-free buffer. See Koziel et al., 2018. J Chromatogr B Analyt Technol Biomed Life Sci. 2018 Aug 1;1091:101-107. After elution from the resin, if needed, remaining traces of endotoxin can be removed with the polymixin resin.
Hi Pierre Béguin, does it matter if I use Triton X-100 instead of Triton X-114? Many thanks, Julen.
I don't know. I would suppose that it does. However, the advangtage of Triton X-114 is that you can use UV absorbance tomonitor its disappearance in the wash. Originally, Triton X-114 was chosen because it enables to perform phase extraction: it will form a phase extracting the endotoxin at 37 °C, which bypasses the on-column washing. However the phase extraction is not as efficient and/or incurs significant losses of protein.
Hi Pierre Béguin ,
Which concentration of Triton X114 do you recommend in the Ni affinity-chromatography? Many thanks in advance.
Julen.
I successfully tried washing material adsorbed to a Co2+ Talon IMAC column with buffer containing 0.5 % Triton X-114 (in the cold, since Triton X-114 will for phases at room temperature), followed by Triton-free buffer until disappearance of most of the UV-absorbing material and elution with imidazole
Depending on the nature of your sample buffer, EndoTrap Red or HD might be appropriate. While EndoTrap HD resin has a greater affinity toward LPS and can be regenerated more often but if your buffer is PBS without Ca/Mg enrichment, EndoTrap Red is your best choice.
There is a section on the page below helping you determine which is best for your sample type.
https://ilexlife.com/products/endotrap-red-endotoxin-removal
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