Greetings everyone
I am trying to mix lysates of two interacting proteins.
1. P1-TEV-SUPERFOLDING GFP-HIS (Using superfolded gfp to improve solubility) size should come 70KD
2. P2-uncleavable HIS size 26 KD
keeping salt 150mM NaCl and 200mM Imidazole for 1st column i got the following profile A
Further while dialysis added TEV into it overnight under same buffer condition 50mM tris 150mM nacl ph8
but observed heavy precipitation
Even though the glowing upper band is not visible but i cant see any cut protein as well.
What could be the reason for this precipitation? on running the precipitant also i cant see any different band.
As Ian mentioned the TEV will also not cut well when precipitating, maybe try RT or warmer incubation and then remove tag and TEV on another IMAC column; most all TEV has a His tag. Its possible a slight disruption of the GFP might be needed to cut, low levels of detergent can often help and GFP is quite resistant to global unfolding. There is a lower band in the UV gel for the digest, what size is expected for the cut protein?
Imidazole affects the TEV activity. I remove it by desalting before adding it to cleave my proteins. You can remove it and then try.
Although I am not sure I understand well your gels, It sounds to me that your protein is precipitating upon removal of the Imidazole by dialysis. If that is the case the steric occlusion induced by the aggregation will prevent the TEV from working properly. If you follow Sunil Singh recommendation you will immediately see if that is the problem. Also TEV requires a reducing environment and is commonly used with 1mM DTT. Make sur that you have some DTT in your cleavage buffer.
Try cleaving while still bound to the column. It may be that hyperfolding is interfering with cleavage site.
..it might also be due to the fold of your protein...i had a case where the DTT was needed for the protein actually for it not to oligomerise...you could add 1 or 2 mM DTT throughout the prufication and see if your protein and the DTT benefits from it...
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