I'm trying to purify adiponectin from bovine serum following the same method of using zinc chelating sepharose column. I ordered IMAC sepharose TM Fast flow column (GE healthcare 28-4046-25 AD) and I used 0.1 M zinc chloride solution for charging of the column before use. I got most of adiponectin in the flow through. The zinc packed IMAC sepharose fast flow column is the same as the zinc chelating sepharose column because elution in this column is depend on imidazole according to manufacture instruction, but in this method the Ph is different.
If anyone knows a suitable method to purify adiponectin successfully from bovine or fetal serum, I will be greatful.
Dear Mohamed,
Which pH value you have used for charging your column with zinc Ions, because you have a solubility problem above pH 5.5. For binding your protein you Need a more alkaline pH value, because the interaction to the immobilized zinc is effected by free electron pairs of the Nitrogen Atom within the imidazol ring, therefore this Atom must be de-protonated. I would suggest you dilute bovine Serum in a Phosphate buffer (do not you TRIS buffer which can interact even if weakly with zinc Ions via its amino Group) with pH 8. Ensure that crude extract you will load onto the column has an pH of 8, otherwise tritrate it to pH 8 with NaOH or concentrated Phosphate buffer. Equllibrate your column with the Phosphate buffer pH8 and than load it on the column. You can wash the column with low amount of imidazol and finally elute with 0.5 M imidazol and/or pH-shift to pH 6.
Good luck,
Martin
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