Ion exchange chromatography question - (Nov/23/2006 )

Can I use Tris buffer (pH 8.0) for purification of metallo-protein (pI 9.0) by using cation exchanger column ?

1. If I can't, why ?
2. Or I should try other buffer such as Phosphate or HEPES

Ps. Tris buufer is recommended for anion exchanger column, but not in the list of recommended buffer for cation exchanger column, by Invitrogen.

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anion exchanger are often Q matrices and interfere with HEPES buffer; Tris itself is cationic and should only be used in defensive concentrations for cationic exchanger chromatography; as metalloproteinases are dependending in conformation of bivalent cations, there is to think not to chelate bivalent cations by phosphate buffer; HEPES will do but it´s rather expenisive for cationic column chromatography;

as we do not know exactly your matrix you like to use we can only answer in general;

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Oops.. sorry recommended buffer by Amersham, not Invitrogen.

Thank you very much, kosmodrom. I use HiTrap column (Amersham), that is Q sepharose matrix.

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If your proteins pI value is 9. Then its better to try negative purification
by anion exchange chromatography. since other protein will have negative charge at pH
8 and yr potein wiill have positive.

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q-sepharose is an anion exchanger. the binding buffer pH must be higher than the pI of your protein in order for the protein to bind to the matrix. tris is an effective buffer in the range of ~7.2-9.2, therefore it is not a good buffer to use with q-sepharose and your protein. you could use tris (below pH 8 would be better) with a cation exchanger such as sp-sepharose or cm-sepharose.

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I thought MOPS, HEPES or even acetate is better buffer components for SP or C- columns. Tris will stick to both columns.

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yes, but it can still be used if necessary.

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