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Changing buffer from pH 7.4 to pH 5.9


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#1 mkhemmani

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Posted 30 November 2010 - 02:02 AM

Hello All,

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.
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#2 BioMiha

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Posted 30 November 2010 - 03:51 AM

Why does it seem a bad idea to just add HCl? I don't see the point in using PBS pH 5 to lower the pH of the Ringer's solution, because you would just dilute the Ringer's and end up with Ringer's/PBS solution pH 5. I think you have to adjust the pH when making the Ringer's solution anyway.

#3 perneseblue

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Posted 30 November 2010 - 09:11 PM

The buffering range of HEPES is somewhere between pH 6.88.2. Outside this range HEPES is either fully protonated or fully deprotonated. In either of these two states, HEPES stops working as a buffer.

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
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#4 hobglobin

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Posted 01 December 2010 - 08:14 AM

Have a look here. Roche's Lab FAQs no 6 (preparing buffers and media) has some tables with buffers and their pH ranges. Perhaps you find an appropriate one.

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