medtjm at bimcore.emory.edu
Mon Aug 19 12:18:40 EST 1996
Mark Prescott wrote:
> I am thinking of trying the Ni-NTA alkaline phosphatase conjugate
> marketed by Quiagen for the detection of HISx6. I expect that this
> product is OK for detecting proteins overexpressed in E. coli but has
> anybody attempted to use it in a more demanding situation (eg. for the
> detection of proteins not over expressed) in yeast or mammalian cells
> with good results.
> Thanks for any feedback.
We tagged a cell surface receptor protein and transfected it into COS-7
cells. By western blot, we could not detect it with either the
Ni-conjugate or MRGSHis6 antibody (Qiagen). We could detect the
recombinant protein by an independent assay, however. We had a
His-tagged, purified cyclophilin prepared by overexpression in bacteria
on the same blot. Memory is dim, but I believe it was detectable only in
the low ug/high ng range using the conjugate. Clearly, one won't achieve
that level of expression in mammalian cells.
Bottom line, I don't think it will work for you very well, if I
understand your plans. Our route around this is to use another tag (now
trying HA, others have recommended FLAG). These are built into the
protein already His-tagged, so we can use Ni-resin to precipitate, then
detect on Westerns with antibodies against the HA (or FLAG) tags.
Hope this saves you some time.
/////////////////////////////////////////////////////////////////////////T.J. Murphy, Ph.D.
Department of Pharmacology 404-727-2467
Emory University School of Medicine 404-727-0365 (fax)
5031 O.W. Rollins Research Building medtjm at bimcore.emory.edu
Atlanta, GA 30322
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