This tag is very hard to release, as it forms an integral part of the
RNAse A. It maybe released with 2M NaSCN but this often results in
denaturation of the carrier protein. This is why the manufacturer strongly
recommends having cleavage site(s) engineered in between the tag and the
The low capacity of the column is also not surprising since the affinity
element is the entire RNAseS protein. While it is not a large protein,
it's still considerably larger than the tiny NTA, IDA, or other
metal-chelating group used to make IMAC resins.
I've used S-tag to purify milligram quantities of proteins but inevitably
engineered these constructs away from the tag in the end, for convenience
reasons. If you know that your protein cannot be expressed with other
tags, then your best bet would be to engineer a cleavage site between the
S-tag and the protein of interest (although even this can potentially
screw up expression). If all else fails, you can try using mutagenized
S-tag - there is a report out there describing rational mutagenesis of the
peptide sequence to reduce the affinity of the S-peptide for the
S-protein. Thus, you may be able to elute with lower concntrations of
> I have a membrane protein with a C-terminal S-tag, and there is no
> site for the S-tag (novagen, e.g, thrombin, EK). I wonder whether I still
> can use this for purification without cleavage of the S-tag. Here are my
> 1) How to release the protein bound to S-tag resin? I noticed they suggest
> 3M NaSCN, 3M MgCl2 or 0.5 M Citrate pH2, but these sound crazy for a
> membrane protein. Does anyone have experience with this?
> 2) It seems that S-tag resin has lower binding capacity than does Ni
> Did anyone ever use S-tag resin for purification of at least 10 mg
> I would probably put a his tag on my protein for purification later.
> Many thanks for any suggestions.