Problems with ion-exchange chromatography..

[nashnash]

Hi

I'm trying to purify my protein using ion exchange chromatography (Q-sepharose, 10mM phosphate buffer pH 7.5). Around 70% of my protein comes out in washing fraction, while only the rest elutes with 0.1M NaCl. My guide says a fresh column behaves like that and the protein will bind more as the column grows old. Is that the case? Or any other suggestions?

(Partial purification has been tried already for my protein with DEAE-Sephadex A-50 column, and it has been found to bind at pH 7. So pH should not be the problem, i guess)

[danko]

Hi Nashnash,

You write the pH (referring to 7.5) shouldn’t be a problem and it is not. Anyway for the 30 % part of your protein mixture. The rest will need somewhat higher pH – say 8 – 9. The stationary phase (quaternary amine, if I’m not mistaken and thus a strong exchanger) will easily be able to handle a higher pH value.
Even the 30 % that are retained are not significantly retained and you can see that by looking at the ion strength needed for eluting it (i.e. 0.1 M). Well retained proteins typically need 0.2 – 0.4 M NaCl to be eluted.

And now to:

[nashnash wrote]My guide says a fresh column behaves like that and the protein will bind more as the column grows old. Is that the case? Or any other suggestions?[/quote]
Yeah I wish And what would be the technical explanation of that phenomenon?

So, here is a quick suggestion: prepare a 0.02 M TRIS, pH 8.3 instead of the phosphate buffer and proceed with everything else unchanged. I would prepare myself for the need of higher NaCl concentration if I were you.

Best Regards

[sabine]

Hi!

My guide says a fresh column behaves like that and the protein will bind more as the column grows old.

I have never heard of nor experienced something like this with the ion exchange resin we use in pilot/large scale production. Q Sepharose is one of those resins...

If I understood correctly: you equilibrate the column with the buffer mentioned, then load the column (do you use the same buffer for the column load?), then wash the column with that phosphate buffer (then you loose 70%), finally you elute the remaining 30% with the same buffer (containing NaCl).

I would simply say, the wash buffer is not suitable. Either its conductivity is too high (very unlikely, 10 mM is really low) or the ph is not correct.

So some questions to think about would be:

- are you absolutely sure that your protein is negatively charged at that pH? Otherwise it will not bind correctly. What is its pI?
- do you loose your protein only during washing or is there some loss in the flowthrough during loading, as well?
- what sort of guide do you use? GE HC offers pretty good downloadable guides for their resins and on the different separation techniques.

Ah, danko was a bit faster now!
At least we have the same opinion...

[nashnash]

I equilibrate the column with 10mM phosphate buffer pH 7.6, then load the sample (in same buffer). Followed by washing with the same buffer and elution with 0.1M NaCl.

There has been a previous study (partial purification) where the same protein has bound to DEAE-Sephadex A 50 using 10mM phosphate buffer pH 6.5 (and eluted with a gradient of 0-0.1M NaCl), so im pretty sure pH 7.6 or 8 should be ok.. And I dont lose any protein in the loading flowthrough, only in 3 fractions of washing (5 ml each). With 0.1M NaCl, the protein elutes after 1 column volume of elution buffer has passed through the column.

I'm rather new to protein purification, so do u feel i might have made some other error that might cause such a problem? Most of the proteins in the sample have bound to the column (visibly) but my protein elutes mostly during washing stage..

[danko]

There could be loading capacity differences. After all, you’re talking about two completely different columns/stationary phases. Your target protein is probably less charged than the proteins that exhibit stronger attraction and thus “looses the competitionâ€

[nashnash]

Hi

thnks for the reply:) I was thinking the same thing, that my protein might be less charged and "lose the competition". I am planning to
1) increase pH to 8.3 and use Tris buffer
2) collect whatever washing fractions I get (containing my protein), and reload it on the same column after removing other proteins that have bound to the column in the first place.
Hope I will be able to purify my protein then!

Also, DEAE-sephadex combines ion-exchange with size exclusion for protein separation, if i'm right. Do you think that could be an advantage over Q-sepharose in my case?

[danko]

It depends very much on the proteins’ charges in combination with their sizes. I’m of the following conviction: One should start off with a simple but well founded methodology and work towards understanding and exploiting the properties of the compound/s of interest. If everything in this context fails, then one could move on towards more exotic and complicated techniques. In the process of the initial tests/phase one learns a lot of things about the compound/s of interest and that makes it so much easier to find the smartest solution.

Best Regards