vectors and his-tags - (Jul/22/2010 )
Just a query - I was having problems purifying a his-tagged protein on a nickel column.
It didn't seem to elute regardless of pH or concentration. Even when NaCl was added in increasing concentrations still nothing.
We figured it was something to do with maybe the his-tag being hidden in the protein or possibly something down those lines.
Eventually I purified it using IEX.
What i'm wondering is we had the protein in the p-QE60 vector and another study had it in the p-ET21a vector. The study with the same protein in the p-ET21a vector had no problem eluting it from the column with imidazole.
Can vectors play a big role in the purification process?
If the fusion tags are identical, the vector doesn't matter. If the tag expressed is different, perhaps a different number of histidine residues or different leading or trailing amino acids, then there can be differences. Also, whether the tag is C- or N-terminal can play a role. More likely, the difference was in the column used, and perhaps the metal -- nickel vs cobalt, NTA (nitriloacetic acid) vs IDA (iminodiacetic acid), etc.
Are you sure that your protein binds to the column? Or can you detect it in the flow through of your column?
Both Studies used NTA columns to purify.Though one study is using a quiagen kit to purify whilst we were using columns which we packed ourselves and ran the purification using an econpump. The differences are that one study has the protein in Pet-21a and the other has it in pQE-60.
I'm not sure if the fusion sites are different - I can't seem to find out?
Benita - it was a bit odd.. A little would be detected on the resin and a little would be detected in the flow through.
Thanks for the help!