Nikola Wenta via (by Nikola.Wenta from
Tue Feb 7 12:33:12 EST 2012

> Hi all,
> I am trying to replicate the experiments of an ex-colleagues whose
> notes require '50mM tris-HEPES buffer (ph7.5)'. I am not sure whether
> the 50mM refers to the tris or the HEPES and was wondering whether

I guess it is achieved by titrating a 50 mM unbuffered TRIS solution against a 50 mM unbuffered HEPES solution (or the other way around) until pH is 7.5. The molarity actually only corresponds to the buffer capacity of the buffer, so the initial molarity of the "stock" buffer shouldn't matter as long as the effective final concentration of the buffer is not too low, i.e. the buffer has lost its buffer capacity. This implies that the colleague initially actually prepared a 500 mM TRIS-HEPES buffer with pH 7.5.
Just a thought: usually pH of TRIS would be adjusted with acid (HCl), while for HEPES a base (NaOH) would be used.

> There is plenty of ambiguity there but I'd think that it means that
> the buffer is made with 25 mM of Tris and 25 mM of HEPES.
> At least that's how it works in our lab: a "1.0 M MES-Acetate"
> is 0.5 M of each component.

I don't agree with DK's statement. Mixing lower molarities of buffers usually yields even lower molarities == dilution.

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