Adam,
We developed a protocol (loosely based on a few previous literature
reports) for metallo-substitution of beta-carbonic anhydrase (a
zinc-metalloenzyme) with cobalt(II). The metal ion in this enzyme is
extremely refractory toward extraction with chelators, and the protein
will not denatu
Hi Adam,
I have not read all the thread as it came all at once and late (9:00pm
here).
I believe the best way to strip a protein of metals is to first adsorb
it onto a solid support (e.g. IEX) and then use a sufficiently low-pH
(say equal or below 6) buffer that contains also EDTA.
You will pr
Thank you everyone for the comments and suggestions. To answer a few questions:
-I do not use a treated buffer system. I have just used the nano-pure water. I
have looked into Chelex, but before I bought it I wanted to see if you all
recommended it. I was trying to avoid this, but it may not be
The answer depends on a number of questions:
* What metal ion are you trying to eliminate?
* What kind of metal-binding site is involved?
o A peripheral or loose binding site? (e.g. surface calcium
ions)--these may respond to chelators
o An active site coordinated metal? (e.g.,
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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 chop
Are you treating all your buffers with metal chelating resin? Are you
washing all plasticware with EDTA and metal-free buffer prior to use? How
are you quantifying your metal content, and what metal ions are
contaminating your sample? You might be pulling out the metal ions, but
they get right b
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 h
