Removal of endotoxin from proteins & DNA - Protein & DNA purification (Jan/31/2005 )
Hello all,
I am new to BioForum. I am looking for protocol(s) for removal of endotoxin from recombinant proteins and DNA using Triton X-114 & Polymyxin B . I would appreciate if anyone could post these protocols on the board.
Thanks,
Devender
Triton X-114:
My protocol is based on that of Kei Yasuda, Yoshiyuki Ogawa, Ikuko Yamane, Makiya Nishikawa and Yoshinobu Takakura, 2004
* Add triton X-114 to DNA, on ice, to a final concentration of 1% v/v.
* Vortex at high speed 10 seconds.
* Heat to 55°C for 15 minutes.
* Centrifuge 20 minutes at room temp
* Take supernatant to new tube, chill on ice
repeat 3 times.
My protocol is based on that of Kei Yasuda, Yoshiyuki Ogawa, Ikuko Yamane, Makiya Nishikawa and Yoshinobu Takakura, 2004
* Add triton X-114 to DNA, on ice, to a final concentration of 1% v/v.
* Vortex at high speed 10 seconds.
* Heat to 55°C for 15 minutes.
* Centrifuge 20 minutes at room temp
* Take supernatant to new tube, chill on ice
repeat 3 times.
Dear friend , would you please give me the reference for the protocol?
Thanks
Hello,
you can also use EndoTrap – it based on affinity chromatography, the binding ligand is a protein.
Please consider that Trition X-114 is not suitable when your desired application is cell culture or animal model.
The problem with Polymyxin B is that it is an antibiotic. The submitted regeneration substance is DOC, what is also not suitable for cell culture or animal model.
More information regarding EndoTrap, the endotoxin removal product from Profos AG, you find here: www.endotrap.de
you can also use EndoTrap – it based on affinity chromatography, the binding ligand is a protein.
Please consider that Trition X-114 is not suitable when your desired application is cell culture or animal model.
The problem with Polymyxin B is that it is an antibiotic. The submitted regeneration substance is DOC, what is also not suitable for cell culture or animal model.
More information regarding EndoTrap, the endotoxin removal product from Profos AG, you find here: www.endotrap.de
I've always used miniprep DNA purified with the triton-X-114 method in my mammalian transfections, and never had a problem. I have also noticed several papers where it has been used to remove contaminants from preparations added to cell culture--so this statement doesnt' seem to be true. Maybe if you aren't careful with the extraction, you might get a little triton left behind, but I've never seen that problem.
Also, I used endotrap (don't remember which one was for DNA I think blue) to remove endotoxins from my DNA preps and I LOST ALL DNA I DON'T KNOW WHERE. I checked everything coming out of the column and didn't get anything. I lost around 50 mg of DNA using this kit. May be it is good for protein but from my experience not for DNA.
I prefer using endo free purification kit from Sigma or Qiagen.
I prefer using endo free purification kit from Sigma or Qiagen.
EndoTrap blue IS suitable for DNA!!! For example, please look in following paper:
INFECTION AND IMMUNITY, June 2005, p. 3686–3692, Luyer et al.
Lipopolysaccharide (LPS) was removed from DNA preparations using Endotrap blue 5/1 (Profos, Regensburg, Germany), after which both preparations contained less than 1 pg LPS per µg DNA.
1 pg/µg regards to 0.001 EU/µg whereas Qiagen said that "Endotoxins efficiently reduced to less than 0.1 EU/ug DNA"
EndoTrap red is NOT suitable for DNA. When you LOST all of your DNA sample I have unfortunately assume, that you have used EndoTrap RED. Profos do not recommend EndoTrap red for DNA. Have you bought your EndoTrap kit from Profos???
Further I want to make you attentive that you get technical support from Profos directly. www.profos.de
I'm pretty sure I used the one says good for DNA which I assumed blue endorap in my post.
So I kindly want to ask you to use our technical support (endotrap@profos.de).
Yours Sincerely,
Stephanie
Sure.
Thank you