Dear all,
would it make sense to include Triton X-100 in the washing step during Ni-affinity chromatography of E.coli lysate in order to clear out endotoxins?
Normally this procedure is only applied in the case of proteins targeted for medical use, but could endotoxin removal also be benefitial for purification in structural biology? I am especially thinking about binding sites or interaction sites that might be obscured by endotoxins or the possible rise of artifacts in structural assays due to effects caused by endotoxins acting as "silent"/unwnated co-factors/ligands?
I would appreciate any advice on this topic. :)
best regards,
Peter
Hi Peter
Triton X-100 is notoriously difficult to get rid of. It depends on your intended use of the His-tagged protein. The best solution would be if you had an assay for the presence of endotoxin to test your preparations. Triton X-114 is also used (Yasuda et al., 2004). If you have a re-foldable protein that is suspected to bind lipid, unfolding, washing and refolding could be an option. In this case, your tag can come in handy.
As a footnote, we have never seen any evidence of the presence of endotoxins in our structural studies (NMR, X-ray). But absence of evidence is not evidence of absence.
The extent of endotoxin removal that is necessary for biomedical applications is overkill in your case. I would think that an additional anion exchange step or a phase extraction step with Tx114 as mentioned above by Kurt would be enough to ease your concerns.
Unless you have some reason to suspect that your protein binds lipopolysaccharide, or plan to inject your protein into someone, special consideration to endotoxin removal should not be necessary.
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