Changing buffer from pH 7.4 to pH 5.9
Posted 30 November 2010 - 02:02 AM
I'm trying to move the pH of Ringers buffer from pH 7.4 down to around 5 to activate my HEK cells expressing a TRPV1 cation channel. My problem is that the buffer range of Ringers is going to be between pH6-8 (being that it's buffered with HEPES (10mM)). Simply adding straight HCl just doesn't seem like a good idea, so I'm wondering what would be the appropriate strategy to bring the buffer to a low pH in a controlled manner. I know that PBS has a pretty wide buffering range and that it can go to as low as pH3-4. So my plan is to make a 100mM PBS at say pH 5 in order to drive the pH down. Does this sound appropriate or would a different type of buffer be required? Any help would be appreciated.
Posted 30 November 2010 - 03:51 AM
Posted 30 November 2010 - 09:11 PM
Thus is a bad idea to add HCl to a HEPES buffered solution to attain pH5. At this pH, there is effectively no buffer in the solution, so any small variation in H+ concentration will lead to large changes in pH.
I am not sure that phosphate buffer can reach pH 5. Citrate or citrate phosphate buffer would be a better candidate.
And instead of adding a second buffer to HEPES ringers solution, it would be easier to reformulate the Ringers solution with this new buffer (substituting HEPES). It would be quite a tedious calculation to obtain pH 5 from a mixture of the initial HEPES ringers solution and the second buffer.
I am not sure, if citrate is suitable for your experiment, other organic acids are available
Posted 01 December 2010 - 08:14 AM
One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.