Detecting HISx6

[TJ Murphy]

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.
> Mark
> 

Mark-

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.
Assistant Professor
Department of Pharmacology                        404-727-2467
Emory University School of Medicine         404-727-0365 (fax)
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