Thanks, DK, for your good advice on rTEV. I suspected as much regarding
the cysteine and imidAzole. Regarding the concentration of imidazole, I
thought 1M was a lot too, but that is what the resin manufacturer and
our plasmid donor have in their protocols. They use Novagen's His Bind
resin for his tag removal. The elution buffer is 0.5M. Our donor uses
bind (or lysis), bind and elution buffers with added 10% glycerol as
well, but they do not add it to dialysis (?). I did, but it still
precipitated. My PI wants me to try concentrating in elution buffer by
stirred cell, and then dialyzing. I wanted to clarify the DTT: you mean
use TCEP instead of DTT, or the other way around?
Thanks,
Vicky
