-----BEGIN PGP SIGNED MESSAGE----- Hash: SHA1 Dear Adam,
- - many plasmids for His-tags contain a cleavage site to remove the tag. In fact this provides you with an additional purifiction step which is complementary to the first Ni-column and therefore quite a good strategy (in addition to chopping off the chelating His-tag). - - you can try other Titriplexes like EGTA, EDTA is only one of a series - - if only the His-tag binds the ion, wouldn't this qualify the protein as 'apo'? If the His-tag is ordered, you could actually replace the ion with Co and use this for phasing, in case this is an issue. Best, Tim On 05/19/2014 07:20 PM, SUBSCRIBE CCP4BB Adam Brummett wrote: > Hello All, > > I apologize for the non-crystal related question. I am trying to > get a fully metal-free apo enzyme. The 6x His construct is > consistently purified with some metal (20-30%). I have attempted > chelating away the metal with up to 30 mM EDTA and DFO and then > dialyzing it away, but this has shown little to no effect. Any > thoughts or recommendations would be greatly appreciated. Thanks. > > Adam > - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -----BEGIN PGP SIGNATURE----- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Icedove - http://www.enigmail.net/ iD8DBQFTekO/UxlJ7aRr7hoRAkhUAKCXbpCx2x4arUbTDtnpDdviIoL3/QCeP+ik aaDa+rmp1AALJhdsDn8els0= =hfzc -----END PGP SIGNATURE-----
