Hi Rafael,
In this case I recommend the use of Zn-charged Chelating Sepharose™ Fast
Flow (Cytiva), as we have established it in the early days when first
applying IMAC to the purification of native antibody fragments (see
PMID: 1367302 and PMID: 8163179).
While the Zn-IDA matrix has slightly lower affinity towards the His6-tag
than Ni-NTA it provides better selectivity and we still use it today for
purifying sensitive proteins including the presence of reducing agents.
Importantly, in contrast to Ni(II), Zn(II) is essentially
redox-inactive, so it does not catalyze the oxidation of thiol groups
and just forms reversible complexes. Furthermore, Zn(II) is non-toxic
and does not cause allergic reactions.
Cheers, Arne
Am 31.10.23 um 23:05 schrieb Thomas Edwards:
Hi Rafael, Dom et al,
Indeed nickel eluted with imidazole stays on all your proteins with a
His tag, and addition of DTT will make almost all proteins go brown
and crap out at this point. But, as Dom says, addition of EDTA
*before* the DTT will solve the problem. Most of the time…
If your protein goes brown after a nickel column and you thought it
was something special to your protein, try again..!
If your protein can’t handle EDTA, try the pH trick suggested in
another post.
If none of that works, then move to GST or some such…
*/Ed/*
https://www.ularkin.org/team-member/thomas-edwards/
Sent from my iPhone. On the run…
On 31 Oct 2023, at 16:48, Dom Bellini <[email protected]> wrote:
Hi Rafael,
Once I inherited a protocol with a problem similar to yours and they
told me that the precipitation was caused by nickel leaking out
during the elution with 250 mM imidazole. I am not sure whether this
was true, however, their fix was to place something like 20 ul of 200
mM EDTA at the bottom of the collection tubes into which the protein
was eluting. This indeed kept the protein soluble, at least long
enough to dialyse or gel filtration.
Good luck!
D
On 31 Oct 2023, at 19:21, Rafael Marques
<[email protected]> wrote:
CAUTION: This email originated from outside of the LMB.
Do not click links or open attachments unless you recognize the
sender and know the content is safe.
*[email protected].*
Hi everyone,
I have been looking on this bb and other websites as well but I
could not find a veredict. We are suspecting that when I elute my
sample from my Ni-NTA column, the imidazole concentration (250 mM)
is making it to precipitate. Once my sample has a cleavable TEV
site, I was planning to incubate my loaded resin overnight with TEV
and get my sample back simply using my lysis buffer. And here lies
the problem. Most of the TEVs are kept in EDTA and DTT and I wonder
if they are essential for its protease activity or if I could use
another reducing agent more compatible with my resin (or maybe do
not add both). I saw that someone did not have EDTA and used
b-mercap. instead of DTT. May I have your comments if you guys
already faced a similar situation?
Best wishes
______________________________________________________
Rafael Marques da Silva
PhD Student – Structural Biology
University of Leicester
Mestrando em Física Biomolecular
Universidade de São Paulo
Bacharel em Ciências Biológicas
Universidade Federal de São Carlos
phone: +55 16 99766-0021
/ "A sorte acompanha uma mente bem treinada"/
/________________________________________________/
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