Protein Expression

Klaus Lun klaus.lun at urz.uni-heidelberg.de
Tue Feb 18 12:50:11 EST 1997


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 
washing.
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



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