His-tag protein purification
Posted 16 March 2005 - 08:38 PM
I am a rookie for protein purification and I am doing his-tag protein purification. My protein is around 26.1kDa and PI is 7.1. After purification, I ran SDS PAGE and found that my protein is in the cell pellet, not in liquid part. I expressed the protein at room temperature for 4 hour with 1mM IPTG. Could you let me know how to solve this problem, please? I have worked for more than 7 months and I am very discouraged.
Thank you so much in advance.
Posted 21 March 2005 - 06:06 AM
If you got to have your protein in solution you could try lysing the cells with 8M urea.
Edited by Linnea, 21 March 2005 - 06:13 AM.
Posted 22 March 2005 - 06:54 AM
Posted 23 March 2005 - 02:25 AM
The first strategy to solve the problem would be to try avoiding the formation of inclusion bodies by slowering the overexpression. Grow the cells at 30C,25C,and 20C. harvest cells at 30mins interval after induction and check for activity in the soluble form. If these tricks dont yeild try subcloning inot a less powerful expression vector.
If nothing works then go for solubilization of inclsuion bodies. This works well with some proteins but not always. There are many aggregate solubilization solutions available as a kit from Hampton Research.
Posted 26 March 2005 - 08:50 AM