I would like to keep high salt concentration during TEV cleavage. Should I expect lower activity of TEV at 1M NaCl? The buffer contains also 50mM Tris pH 7.5, 1mM TCEP and 300mM imidazole.
Best
Hi there,
According to this paper: http://www.ncbi.nlm.nih.gov/pubmed/7793070/
TEV is fully active in the presence of NaCl up to 2M.
My experience is use low salt or TEV is very inactive. The lower the better, even adding it after dialysis into low salt buffer increases yield of cleaved protein. It also is quite inactive in Imidazole as well so best to get rid of that as well.
Hi
As You can see TEV activity was not affected by 1M NaCl. The cleavage with TEV (3mg/1l of cell culture) occurred overnight in cold room during dialysis to remove 300mM imidazole.
1. Before TEV
2. After TEV
Best
Leszek
Hi leszek,
Can you elaborate more about your protocol, I am going to do the same with TEV?
Thanks
Source of a cheap nuclease is here: http://www.protean.cz/en/recombinant-protein/11/endonuclease-s-marcescens
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