ffer conditions.
Good luck!
Irwin
From: CCP4 bulletin board on behalf of Hughes, Jonathan
Sent: 02 November 2023 09:17:30
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] AW: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage
(this is from a different jon)
if a chelator real
23 19:53
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage
I have always performed TEV cleavage after eluting my protein from the Ni-NTA
column (TEV cleavage performed during overnight dialysis in imidazole free
buffer as imidazole inhibits TEV activity at
I have always performed TEV cleavage after eluting my protein from the
Ni-NTA column (TEV cleavage performed during overnight dialysis in
imidazole free buffer as imidazole inhibits TEV activity at high
concentrations). I use TCEP as reducing agent and have never included EDTA,
by this method I've
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
Dear Rafael
In addition to the excellent suggestions already offered by previous
responders, I can attest that Ni-penta resin (sold among others by a
company with an odd name Marvelgent) is resistant to EDTA and DTT, and it
leaks very little Ni (almost none). Plus, it elutes with low inidazole,
ow
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 protei
n, UK
==
about.me/david_briggs<http://about.me/david_briggs>
From: CCP4 bulletin board on behalf of Rafael Marques
Sent: Tuesday, October 31, 2023 7:21:21 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage
External Sender:
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 t
Hi Rafael,
the simple answer is that the EDTA and DTT( or any other reducing
agent) is not required for the TEV activity.
You can have your TEV protease in 20 mM Tris pH 7.5 (RT) and 150 mM
NaCl, plus either 10% Glycerol or 50% glycerol depending on storage
conditions preference - 80 or -20C respec
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
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