'Sticky' His-tag proteins

Ian McFarlane i.mcfarlane at icrf.icnet.uk
Fri Jul 24 08:33:54 EST 1998


Is your protein stable in solution? It may have some hydrophobic patches
that a bit of non-ionic detergent may block. I take it that you have
boiled the beads in SDS-PAGE sample buffer to show that your protein is
still attached. One other possibility is that the protein is not stable in
its purified form. APP for instance falls out of solution never to return
even in sample buffer, DTT (1mM) may help this but you can only add it
after the column (you can use up to 20mM b-ME). Do you have the same
problem with the denatured protein perhaps you can renature it after it is
pure?

HTH

Ian Mc

Boro for the cup! Eventually!





In article <35B85720.8685CBEA at leonardo.ls.huji.ac.il>,
yoramg2 at LEONARDO.LS.HUJI.AC.IL (Yoram Gerchman) wrote:

> Hi
> 
> >Has anyone experienced a 'sticking' of His-tag proteins to
> >agarose/Sepharose/any sort of beads at all, causing problems with
> >purification (even NTA-agaroes beads that have been stripped of Ni++
> with
> >EDTA). Ideally I want to keep things 'native'. Phararmacia suggested
> using
> >a borate buffer to reduce non-specific interactions but this didn't
> seem to
> >help.
> 
> >Any suggestions would be greatly appreciated
> 
> >Rob Layfield
> >University of Nottingham
> 
> I'm not sure if it will help but we purify our protein in the present of
> 500mM NaCl. It might "shade off" some charges in protein/agaros.



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