Tris-HEPES

[Yvonne Couch]

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 anyone had any experience with this? I am using it as an extraction buffer for fresh tissue (the other components of which are sucrose and EDTA) and need to maintain the integrity of the enzymes within the tissue, rather than the cells. If anyone has any suggestions for a replacement for this buffer or any more details on how to make a tris-HEPES buffer, I would be most grateful.
Regards
Yvonne

[Nikola Wenta]

> 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.
Cheers,
Niko

p.s.:
> 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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[Dr Engelbert Buxbaum]

In article <mailman.222.13285...@net.bio.net>,
yvonne...@pharm.ox.ac.uk says...

>
> Hi all,
> I am trying to replicate the experiments of an ex-colleagues whose
> notes require '50mM tris-HEPES buffer (ph7.5)'.

This is very ambiguous, I would use 50 mmol (6.06 g) Tris (as it stands
closest to the concentration, so presumably that is what it refers to),
dissolve it in about 800 ml of water, add any other components required,
titrate with HEPES to pH 7.5 and then make the volume to 1 l.

In a publication I would then write 50 mM Tris, titrated to pH 7.5 with
HEPES, which would avoid any ambiguity.

I would, however, also predict that it makes zilch difference how you
interpret this statement, as long as you have a reasonably well buffered
solution reasonably close to neutral pH, things are likely to work.
Having both the cation and the anion buffer at the desired pH increases
the buffering capacity without increase in ionic strength. High solute
concentrations reduce enzymatic activity as solutes bind water that
enzymes require as a "grease" during conformational changes.

One warning, though: If you plan on doing ion exchange chromatography,
use only one buffering ion, and the one that is not bound to the column
(that is, tris on an anion and HEPES on a cation exchange column).
Otherwise, the pH on the column may change significantly during the run,
exposing your enzyme to potentially harmful conditions.