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Should I worry about endotoxin? - (Apr/27/2011 )

I'm highly inexperienced, so any help would be appreciated.

I'm purifying a large amount of 6xHis tagged protein expressed in BL21 (DE3) using an NiNTA column. Ultimately the protein is going to be injected into mouse eyes to check for vascularization (I don't know the specific details of this).

Do I need to worry about removing endotoxin using something like detoxi-gel, or should the NiNTA column take care of it?

Thanks.

-Aallea-

Hola this is only an opinion, not an expert recommendation, Im not expert in something, probably in the purification process you have eliminated them mainly in the washes before elution, but you have to do an assay. Here we inject in mice in blood new molecules and when they die are diying with oral administration too probably by the product toxicity. For generation of antibodies, in the past, we have injected purified proteins from bacteria or baculovirus in rabbits or mice or directly extracted from a PAGE and nothing happened.And thrid, think in people who injected drugs in the street without any sterility, they could be sick by any infection. So I would filter the protein solution by a 0,22 um filter and make a try. Wait for any expert opinion if you want. Buenasuerte

-protolder-

Buena suerte

-protolder-

protolder on Thu Apr 28 05:51:53 2011 said:


Buena suerte


Thanks, I really appreciate it.

-Aallea-

Yes, you should. LPS higly activates the endothelium and is present in any preparation of recombinant protein from bacteria. This might not be such a problem when the protein is applied on the eye. However, if you inect it in the eye, measure the level of endotoxin. There are kits available to easily measure the level of endotoxins. I f you work in medical research, somebody around you has certainly that type of item.
All the best

-redbio-