Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

[Irwin Selvam]

Hi,

In my hands, I have found the addition of TCEP (~0.5 mM) helps with TEV 
cleavage efficiency, especially reactions at 4C where TEV is much less 
efficient anyway. The caveat is that my targets may have been "happier" with 
TCEP present, therefore encouraging more efficient cleavage. The variance in 
what you've been able to find online, and reflected in the responses thus far, 
could be down to different labs using different TEV constructs. Older 
constructs seem to be more sensitive to reducing agent presence whereas 
optimised versions of TEV are much more efficient so any drop off is less 
noticeable. It may be worth looking at your cleavage buffer composition too - 
not too much salt, glycerol, detergents etc

As for on-column TEV cleavage, if your TEV is also His-tagged then it will 
likely be less efficient. If you're willing to try a new construct, I have had 
great success with on-column cleavage using target bound to Streptactin XT 
resin. If you want to stick to Ni-based IMAC, Cytiva's Ni Excel, BioRad's 
Profinity and Protein Ark's Fastback Ni Advance all leach less than 
conventional Ni-NTA/IDA under less than ideal buffer 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 really is necessary at this stage (post AmS?), in any case use 
IDA instead of EDTA! that's pretty obvious from the affinities of EDTA/NTA/IDA.
cheers
jon

Von: CCP4 bulletin board  Im Auftrag von Jonathan Bailey
Gesendet: Mittwoch, 1. November 2023 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 high concentrations). I use TCEP 
as reducing agent and have never included EDTA, by this method I've never had a 
problem with membrane or soluble proteins and get almost 100 % cleavage of the 
tag.

Best,

Jon





On Tue, 31 Oct 2023 at 19:21, Rafael Marques 
mailto:rafael_mmsi...@hotmail.com>> wrote:
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"




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

[ccp4bb] AW: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

[Hughes, Jonathan]

(this is from a different jon)
if a chelator really is necessary at this stage (post AmS?), in any case use 
IDA instead of EDTA! that's pretty obvious from the affinities of EDTA/NTA/IDA.
cheers
jon

Von: CCP4 bulletin board  Im Auftrag von Jonathan Bailey
Gesendet: Mittwoch, 1. November 2023 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 high concentrations). I use TCEP 
as reducing agent and have never included EDTA, by this method I've never had a 
problem with membrane or soluble proteins and get almost 100 % cleavage of the 
tag.

Best,

Jon





On Tue, 31 Oct 2023 at 19:21, Rafael Marques 
mailto:rafael_mmsi...@hotmail.com>> wrote:
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"




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

[Jonathan Bailey]

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 never had a problem with membrane or soluble proteins
and get almost 100 % cleavage of the tag.

Best,

Jon





On Tue, 31 Oct 2023 at 19:21, Rafael Marques 
wrote:

> 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"*
> **
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

[Prof. Dr. Arne Skerra]

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  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 
 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.

*.-owner-ccp...@jiscmail.ac.uk-.*
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"/
//



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 







--
Prof. Dr. Arne Skerra
Lehrstuhl f. Biologische Chemie  |  Technische Universitaet Muenchen
Emil-Erlenmeyer-Forum 5  |  85354 Freising (Weihenstephan)  |  Germany
Phone: +49 8161 71 4351  |  Fax: 4352
eMail: ske...@tum.de  |  http://biologische-chemie.userweb.mwn.de



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

[Artem Evdokimov]

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,
owing to the single available chelation point offered by the immobilized Ni
ions.

Depending on the resin and other conditions tou use, there generally is no
reason to worry about the small amount of DTT and EDTA that may be present
in TEV preparations. On the other hand, there is a reason to be slightly
concerned that the leaky Ni ions may inhibit your TEV.

In summary, you have to try it on small scale, the odds are good that
cleavage on resin will work for you. Notably it even works when TEV itself
is his-tagged, presumably owing ro the dynamic nature of His/Ni
interactions (strictly speaking, resin affinity is only about 1 uM, but
avidity and intra resin exchange and recapture keep the bulk of protein
trapped in the resin).

Good luck!

Artem

On Tue, Oct 31, 2023, 3:21 PM Rafael Marques 
wrote:

> 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"*
> **
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

[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  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  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.
.-owner-ccp...@jiscmail.ac.uk-.

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"




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

[David Briggs]

Hi Rafael,

For completeness -  there are alternatives to imidazole for eluting proteins 
from Ni-NTA resins:

EDTA - (strips Ni2+, and therefore everything bound to the Ni2+) - 50mM should 
be sufficient, in your favourite buffer/salt system. Obviously not appropriate 
for metalloproteins. You'll need to recharge the column/resin afterwards.

Low pH - Citrate or Acetate buffer with a pH lower than 5.5 (lower still for 
multimers) with an appropriate salt concentration. Obviously test this on a 
small scale first to see if your protein tolerates the drop in pH. You can 
reduce the time of exposure to low pH by eluting the protein straight into some 
1M Tris or HEPES to bring the pH back up to something more neutral.

Hth,

Dave


Dr David C. Briggs CSci MRSB

Principal Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, 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: Use caution.

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"




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1

The Francis Crick Institute Limited is a registered charity in England and 
Wales no. 1140062 and a company registered in England and Wales no. 06885462, 
with its registered office at 1 Midland Road London NW1 1AT



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

[Dom Bellini]

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  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.
> .-owner-ccp...@jiscmail.ac.uk-.
> 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"
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

[Nikolay Dobrev]

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 respectively.

Normally you need to take care that you are dilating the TEV several
fold (at least 5) in order to drop the Glycerol concentration below
10% not to have an inhibitory effect on the activity.
Also your on-column cleavage buffer should not contain higher than 300
mM salt, otherwise the TEV activity will be decreased as well.

Go ahead and give it a try.

Good luck!
Kind regards,
Nikolay

On Tue, Oct 31, 2023 at 3:21 PM Rafael Marques
 wrote:
>
> 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"
> 
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

[ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

[Rafael Marques]

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"




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/