I am working with lactoferrin and over my protocol for expression in vitro I have been used urea (7M) buffer for denaturation, however, I would like to proceed an assay with my lactoferrin and it should be intact. Can I reverse denaturation by urea of my lactoferrin? I appreciate any idea.
aletheia
Hi Aletheia,
Of course you can, it doesn't mean it will be easy. The first approach is a direct dialysis against a proper buffer such PBS or similar. According as this results you can think in a step-by-step descending dialysis from 7M to 0M (if protein precipitate). Alternatively you can perform a quick change of buffer using a G25 matrix used normally for desalting.
The other important point to think is if lactoferrin needs iron or not to refold or another co-factor. In which conditions you performed your expression?
I hope this help you!
Oi Aletheia,
Tenho pouca experiencia com proteinas do leite mas acho que uma vez que a lactoferrina foi "denatured" com 7M ureia, eh muito dificil de reverter a denaturaçao. Mas tu podes tentar de dialisar a protéine em membranas ou cassetes de dialise (http://www.piercenet.com/cat/slide-a-lyzer-g2-dialysis-cassettes) mas elas custam caro... Que volume tu obtens apos a expressao in vitro? Se o volume for grande, valhe a pena tentar. Beijos enormes para ti e para tua familia, Noelita
I agree with Diego Sebastian. Rapid refolding of proteins denatured by Urea (up to 8 M) is achievable with buffer exchanging by desalting the solution using a G25 matrix, since your protein is not precipitated. Otherwise, dialysis is a better choice.
Hey all, I performed my assay, and right now I am waiting for the results.
Thank you guys for your reply. It was ver helpful.
Obrigada Noelita, eu estou plotando os dados pra ver se a proteina mantem a atividade. Muito bom ter noticias suas. Bjao amiga :-).
Jorge, amigo, valeu a dica. QQ coisa bato um fio pra ti. A gnt segue aqui com os experimentos. Bjao
- Badges
- Science topic