In article <[email protected]>, [email protected] 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. _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
