I have a protein that I purified in PBS buffer, pH 7. I will do dialysis to remove salt and will then further purify the protein with ion exchange chromatography.

I will need to use another buffer (Tris-HCl) with pH 8 in the chromatography, and so my question is: Can I change the buffer system for my protein in the dialysis step? Or would you recommend me to use the Tris-HCl buffer from the very start and then just adjust the pH for the chromatography?

Thanks in advance!


4 Answers 4


Assuming that your protein can handle being in the new buffer, you have multiple options.

Dialysis will work although you might want to consider using a centrifuge spin filter with a suitable MW cutoff to gradually switch buffer and concentrate the sample. Be aware that some of your protein will stick to the filter, giving a loss of sample.

Alternatively you can perform size exclusion chromatography to switch into the new buffer, but you better be sure that the sample will remain stable or you may clog the column. If you can perform affinity chromatography, you can also opt to change buffer in this step.

You can of course try to start with a Tris based buffer and simply adjust pH by dialysis in a tris buffer with the desired pH. Just make sure that the dialysis volume is large enough to counteract the buffer in the sample.

Instead of using PBS you can also try with TBS (Tris-buffered saline), see wiki for details. https://en.wikipedia.org/wiki/Tris-buffered_saline


There are a number of ways to address this, and the other answers are certainly correct. Another strategy, especially if you already have the sample in PBS, is just to dilute it in the low salt ion exchange buffer. This works because you don’t need to completely remove the phosphate or salt, just get it low enough so that it doesn’t interfere with binding to the column. This method also has the advantage of minimizing the exposure of your protein to low salt, which can destabilize it, and is often used for ion exchange chromatography.

As an aside, if you’ve never done IEC with this protein before, you may want to do a trial run first with a small aliquot of your sample so you can make sure the conditions you’ve decided upon are suitable.

  • 1
    $\begingroup$ Good point, dilution of the sample might be the easiest way forward, if feasible in regards of sample volume and flowrates. $\endgroup$ Jun 30, 2019 at 6:48

Two accepted methods of 'desalting' a protein and/or changing the buffer are dialysis and gel-filtration. Dialysis is time-consuming, and gel-filtration usually requires a concentrated sample (It is difficult to gel-filter a 250 ml sample unless a very large column is available, for example, but dialysis of such a volume is relatively easy).

You do not say whether you are using cation- or anion-exchange chromatography, but note that the charge on the buffering species is positive with Tris, but negative for phosphate, and it is usually desirable that the charge is the same as that of the ion-exchange column, as you do not want the buffering species to be exchanging and possibly exposing your protein to 'local' extremes of pH, especially if your protein is sensitive to pH changes: use a positively charged buffer for anion-exchangers such as DEAE-cellulose, but a negatively-charged buffer for cation-exchangers such as CM-cellulose. (This is the 'strategy' adopted in Chromatofocussing, and I'll bet that is why the protocol requires a change in buffer as well as pH).

Note also that a protein that binds to an anion-exhanger at pH 8 may not bind at pH 7: in Chromatofocussing on an anion-exchange column, for example, we lower the pH to elute the protein.

Ref: Protein Purification. Theory and Practice, R.K. Scopes


Impossible to answer this in detail without knowing why you started with PBS, but simpler is better so if you can start with a buffer that is compatible with the ion exchange step then I would try that.

Dialysis is a reasonable way of changing buffers, but do you know whether your protein is soluble in Tris at pH 8 without salt?

If you have a relatively large sample you might consider doing a test dialysis using a small fraction using something like this kit.


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