Hi there,

I don't think it is a problem as long as the protein is able to interact with the solid phase in the starting condition. If you want to get a uniform system at first then a desalting step on a PD10 using your starting IEC buffer as eluant is very quick and efficient prior IEC.

If MES is used then it is cation exchange chromatography. TRIS is used for anion exchange chromatography. This is to not compete for protein adsorption.

Dear John

i think that the problem is not the buffer that you use but the pH of the buffer.

Are you planning to perform cationic exchange? Because generally MES is used in the range 5-6 of pH for cationic exchange while Tris is more common for anionic exchange at ph=8.

For cationic exchange, if your buffer do not contains NaCl, You can dilute your sample that now is in Tris buffer in large volume of MES buffer (eg 1:10 times) and once the solution reach the pH of the final buffer you can load the sample in your coloumn equilibrated with MES, but of course in this case you will need long time to load the sample because your volume increase a lot.

As Dominique suggest, if your sample do not has big volume a fast and powerfull alternative is desalting using PD-10 gravity coloumn.

You can found a tutorial about buffer exchange by PD-10 on my blog:

http://proteocool.blogspot.com/

ProteoCool n°11(Rapid buffer Exchange by PD -10) at page n°3 (protein purification and characterization)

good luck

Ciao

Manuele

MES is negatively charged, that is, an anion. If you did anion exchange chromatography in the presence of this buffer, MES would bind to the column, this would affect buffering capacity and pH of the solution unpredictably, with possibly negative consequences for your protein. Therefore, for anion exchange chromatography, use a cationic buffer and vice versa.

@Manuele Martinelli,

I have 200ul of protein in 20mM Tris and 50mM NaCl. My target is for it to be in 25mM MES and 40mM NaCl. Would simply adding 1.8ml (1:10) of my target buffer to the 200ul protein suffice? The end volume does not matter as I can concentrate and we have lots of different sized sample loops. Keeping the final salt concentration of 40mM is crucial as the IEX is run on a salt gradient.

Thank you.

with 200ul you can easily run a PD-10 desalting coloumn. You equilibrate tie coloumn with the mes buffer, load the sample and in 20 minutes and the buffer is exchanged.

Give a look to my tutorial

http://proteocool.blogspot.com/

ProteoCool n°11(Rapid buffer Exchange by PD -10) at page n°3 (protein purification and characterization)

good luck

Manuele

If you did that, the NaCl concentration would be slightly higher, 41 mM. There would also be 2 mM Tris left. Whether that is a problem, depends on your application.

Besides desalting columns as mentioned by Manuele Martinelli (although for this small volume I'd prefer NAP5 over PD10) you could also use centrifugal filters or a small Amicon cell for buffer exchange.