Metal chelate chromatography

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JackBean
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Metal chelate chromatography

Post by JackBean » Mon Sep 14, 2009 8:05 pm

Hi all,
is there anybody with experience with metal chelate chromatography (immobilized metal ion affinity chromatography, IMAC).
I wished to try it as one (or more) step(s) in my purification protocol and tried to study in the 2nd edition of Protein purification textbook, but it didn't work very well, or better said, it was kond of confusing:

First of all, I found, that it might be usefull to wash unbound metal ions from the matrice after loading with the elution buffer. As I used the batch method, I tried it. I tried elution by imidazol and amonnium chloride (see below). I would expect, that the unbound fraction of proteins will be the same (as the binding buffer was the same), but it wasn't. In the case of imidazol, all the activity was it unbound fraction and the matrice was nicely clean after elution, in the case of NH4Cl elution, I was not able to recover the activity and also the the matrice remained "dirty" after elution. What could be the reason? Could there stay some imidazol? (But I have equilibrate it twice with loading buffer).

Second, in the book where some examples of applications and in one of them used also the ammonium chloride and in that case, they replaced sodium chloride in loading buffer by ammonium chloride in elution buffer, so I have used the same princip. I was thinking, whether the lower ionic strength (the ammonium ions bind to ions, so the is "deplenition" of ions in buffer), could be responsible for inability of elution? The ammonium ions should not matter the activity measurement (see below).

Third, I have tried Cu, Ni and Zn. In the book, there was several times written, that their binding strength decreases in this order (Cu > Ni > Zn > Co = Ca, but I have used IDA, which should not bind Co or Ca, so I haven't tried them). Anyway, Zn was the only case, where I was able to recover some activity (but not all), so this is proof, that NH4 should not mattwr in the reaction. BUT, I have spill out about half of the matrice before sample binding. Do you think, that the reason of activity recovery could be because of its lower binding strength compared to Cu or Ni or because of higher finnal ammonium concentration (in the normal reaction it was half of concentration in the elution buffer, whereas in this case it was 2/3 of the original concentration).

I hope, you will understand, what I mean :)

Thank you, Tomas
http://www.biolib.cz/en/main/

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