I’m trying to purify a his-tagged protein from baculovirus-infected insect cells.
First time, I just pelleted my cells, washed them with PBS/10% glycerol, pelleted again and lysed with 1M Nacl, 20mM Tris pH 7.5, 10% glycerol, 0.01% NP40 and PIC (my protein is tightly bound to DNA…). This first time, everything just worked fine, amazing expression, and everything was soluble !
Doing it again, it just didn’t work anymore… My protein went from entirely soluble to completely insoluble… I used the same conditions, the same buffer, so I don’t get why it’s not working anymore… I’ve been now trying for 3 months so solubilize it and it’s just impossible… I’ve screened different lysis conditions (urea, higher salt, different detergents, reducing agents, dounce homogenizer, different sonication time, fresh or frozen pellets…).
Do any of you have an idea on what I could try to solubilize this protein ?
Thanks a lot !
Edit : I just want to add that I want to keep my protein native, my aim being to crystalize it... So, refolding could be considered but not as a first choice.
Edited by mgn67, 16 May 2011 - 03:10 AM.