his-tag buffer

Arnoud van Vliet avvliet at ision.nl
Wed Jun 6 16:18:49 EST 2001

> Can anybody tell me why Tris buffer is
> not a good tool for Ni-NTA resine
> washing in protein purification?
> And why phosphate buffer is recomnded?

Might it have to do with buffering capacity at the pH where it is used? I
have mostly used pH to elute protein, and for this one needs a pH of
4.5-5.5, and washing at pH 6. Tris is useless at that pH, as buffering is
almost completely absent. Mostly the protocols have to be able to be used
everywhere, so they might have just chosen phosphate buffer because of the
range of pH values it will buffer to.

A similar thing is used with the Boehringer DIG system. They recommend a
maleic acid buffer pH 7.5 for all their DIF hybridisation applications,
while it hardly buffers at that pH. Tris would be much better, but cannot be
used for RNA work since you cannot treat it with DEPC. So the easiest thing
to do is always recommend the same buffer....

hope this helps

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