I've been trying to purify two full-length proteins (N-His and C-His tagged) for several months, but I keep encountering degradation during the purification process.
On SDS-PAGE, the proteins are clearly expressed in both the crude pellet and supernatant, but as I proceed with purification, I consistently see degraded fragments that are more intense than the full-length band.
I've already attempted to optimize the lysis, wash, and elution buffers, but unfortunately, none of these adjustments have resolved the issue.
Has anyone encountered something similar, or have suggestions for strategies that might help preserve the integrity of these full-length proteins during purification?
Any advice would be greatly appreciated!
Did you purify your protein at cold tempreture? If not, I think you can try it at 4 degrees throughout the whole process
I did all purification on ice but I still experience degradation
Hello, I think you can avoid enzymatic degradation of proteins by adding protease inhibitors to the protein lysis buffer. Additionally, has the purification method for these two full-length proteins been reported in the literature? You could refer to the lysis buffers used in others' papers, which might be helpful to you. Also, could the smaller bands you see on your gel be contaminating proteins or interacting proteins?
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