vectors and his-tags
Posted 22 July 2010 - 03:34 PM
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?
Posted 22 July 2010 - 07:24 PM
Posted 27 July 2010 - 05:23 AM
Posted 11 August 2010 - 03:25 AM
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!