Hello. HEPES-containing buffer is known to be efficient as buffering system within 6.8-8.2 pH range.
Does anyone have experience using a buffer slightly outside this range (e.g. 6.5/8.5 or more for HEPES) for protein assays?
In principle, it shouldn't be drastically deprotonated/protonated. Also I checked the pH of 2 months old buffers with 6.5/8.5, and it just increased/decreased for 0.05, so it seems to be stable. The constituents of assay itself are also neutral, so should not influence on system's pH significantly.
Also I found this: https://www.hopaxfc.com/en/blog/hepes-tris-buffer-and-ph-control where is written that pKa+-1 interval should be OK.
I tried MES and Bis-Tris buffers as alternatives for pH 6.5, but my analysis works worse than with HEPES pH 6.5, that's why I am asking.
Thank you.
The buffering capacity (ability to resist a change in pH) will be very much reduced compared with a pH more centered on the pKa, which can be somewhat countered by increasing the buffer concentration. For the lower pH, I would prefer to use MOPS-NaOH as the buffer. For the higher pH, I would prefer to use Tris-HCl.
it very much depends on what your test necessitates. Hepes at pH 6.5 will be essentially useless to counter the effect on pH of the addition of an acidic substance during your assay.
if you look at the pH titration curve of any buffer, you can estimate that for a buffer solution with pH = pKa, the pH of the solution will be stable as long as the amount of whatever acid/base/titrant/additive you add to it is less than ~10% of the buffer concentration. the further away you go from that point, the less potent your buffer will be, especially in directions away from the pKa.
If you can pH-adjust all of your additives precisely, that's not too huge of a deal.
You say Bis-Tris and MES don't work well. Could you tell us more about the kind of assay you do? Are there divalent ions involved? do you use molecules like ATP, TCEP, or other high-pH-effect elements?
Some typical buffers are not indicated for use with metals...
I will just add that the problem is not with change of pH of a buffer standing on a shelf. That one has no reason to change pH. The problem is, that the buffering capacity will be lower. In principle, you can counter that by increasing concentration of your buffering system.
Ulric has also good point, that it depends on what direction you expect change of your pH. If the pH should increase, than going below the range will be more OK than if it should decrease, because you will approach the pKa and thus making the buffer stronger.
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