Im trying to express a transcription factor in bacterial cells, and i am
using quiagen's pQE-30 vector with an N-terminal 6*His-tag and also
quiagens Ni-NTA resin to purify the protein. The expression seems to
work well, i got a specific protein that binds to the Ni resin (under
native conditions !), but when i want to perform a large scale
isolation, lots od other proteins also eluate from the column after
So, how can i get rid of this proteins ? the manual say trying washing
with 1% TX-100, 1M NaCl, 40 mM Imidazole, 30 % Glycerol, 10 mM
ß-Mercaptoethanol .. ?? The washing is performed in wash-buffer (10%
Glycerol, 0.3 M NaCl, 50 mM Na-P pH5.8).
Should I now add all these components to the wash buffer at the same
time and try a big mix, or test them seperately ?? Can some of the
reagents affect the activty of the protein ?? Has anybody made
experience with this reagents ? I need the protein later for DNA
bandshift assays, so it's native function is essential.
I elute the protein with an 0-0.5 Imidazole gradient in wash-buffer. How
long can the factor be kept in this buffer at 4°C ?? Can I freeze it at
-20°C ? In which conditions can I do a long-tern storage of the active
protein ?? And do i have to get rid of the Imidazole in the elution
buffer ?? Does it interfere with the binding assay ?? and if yes how can
i get rid of the imidazole ??
Please help me --- Thx Klaus