I need a solution for purification of an his-tagged protein

Wolfgang Schechinger novalidaddress at nurfuerspam.de
Tue Apr 25 14:55:27 EST 2006


Dear Bazeille,

How did you elute your protein from the Ni column? Mean, did you elute
with increasing concentrations of imidazole to get as much unspecific /
unwanted stuff off as possible? Then, elute it either with further
increasing imidazole oder just EDTA (then you have to re-charge the
column with eg Nickel chloride or -sulfate)

My suggestion for the workup is a stepwise ammomium sulfate (AS)
precipitation (actually, I had done it before the Ni column as you may
remove a lot of unwanted and interfering stuff without much effort).
You may start with 30% saturation and increase the AS in 5% steps up to
70%. You may add solid AS in order to keep the volume small, but add it
slowly.
You might optimize the procedure with an aliquot if your protein and
analyze the fractions by SDS-PAGE after dialysis and then run the big
prep in batch mode.

Gel filtration is a good or bad idea, depending on the size of your
contaminant(s).
Ion exchange on a mono-Q or DEAE-agarose and elution with a salt (eg
NaCl) or pH gradient might work well then for further cleanup. For
'polishing' then HIC or another affinity step, depending on the
properties of your protein.  

Best, 

Wo



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