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-   -   Apoenzyme reconstitution with bivalent metal cofactors (http://www.molecularstation.com/forum/protein-science/88216-apoenzyme-reconstitution-bivalent-metal-cofactors.html)

kiwi 04-03-2014 01:22 PM

Apoenzyme reconstitution with bivalent metal cofactors
 
Hi there,
I want to investigate the involvement of metal ions in the activity of the enzyme I am working with. I am using EDTA at a final concentration of 100mM to strip any metal ions from the enzyme. I am then using a 20mM Tris-HCl buffer (prepared with deionised water) to do three subsequent rounds of buffer exchange through an amicon ultra filter (with a molecular weight cut off 9KDa below the size of my protein). The EDTA treatment is deactivating the enzyme but I am having issues with the enzyme reactivating after buffer exchange.

I have treated all glassware with nitric acid prior to making up solutions (including HCl used to pH the Tris). However my protein samples because they are such small volumes are always stored in eppendorfs.

I tried treating the tris buffer whith CHELEX 100 resin spinning for 1hr at room temperature but it did not seem to improve the situation.

I used an ultra pure tris powder to make up the tris-HCl buffer and in this case the enzyme was not being reactivated after buffer exchange. However when I tried to repeat it the enzyme was again reactivated after buffer exchange. Could this be because instead of using a fresh amicon column for buffer exchange I used the previously used one which had been stored at 4degrees with 20% EtOH inside?

Can anybody give me some advise, Please?


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