secreted eukaryotic HIS-recombinant protein

Wolfgang Schechinger novalidaddress at nurfuerspam.de
Tue Apr 4 04:09:29 EST 2006


You might precipitate your raw protein from the media and resuspend it
in binding buffer before you go on the column.

Check the papers by Roffler-S, it works like a charm

the ref probably ist this one:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=14725509&query_hl=1&itool=pubmed_docsum

Wolfgang Schechinger
Endocrine Research Lab
BG & University Hospital Bergmannsheil
Bochum, Germany

as wrote:
> Dear NG.
> I am producing HIS-tagged secreted proteins in insect cells. There is a
> N-terminal v5 and poly HIS for purification. However we are not able to bind
> the recombinant protein from the supernatant to nikkel/cobolt columns. Doing
> a western blot (on the N-terminal v5)  I can see that there is plenty of
> protein, but that it runs through the coloumn without binding. We do dialyse
> to get rid of HIS from the media, and we also add NaCl to reduce
> aggregation. I think that the protein might aggregate in the supernatant so
> that it doesn't bind to coloumn.
> Does anyone have suggestions on how to prevent/reduce aggregation if that is
> the problem. We want the native protein with active disulfide bonds, so
> adding for example DTT is not an option! what about Triton for example??
> Thanks in advance!
> A.S



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