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"*
> **
>
> --
>
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[ccp4bb] Help needed finding hit condition

[Jonathan Bailey]

Dear CCP4bb community


I apologies for the slightly off topic post.


We have recently had success crystallizing a membrane protein (diffraction
> 3 Å at a synchrotron source) using the *in meso* method, the hit
condition was from the Jena Bioscience screen Pi-minimal condition number
#57.


Hit condition – 47.1 % w/v PEG1000, 150 mM Tris pH 8.0, 80 mM Potassium
bromide


The screen is old and expired 12/20/2013 (lot # JBS00013133), we have tried
to reproduce the crystals using homemade optimization screens around the
hit condition but have not had any success. We have tried reproducing the
hit using a new (not expired) Pi-minimal screen but had no success. We are
only able to reproduce the crystals using the expired screen and we do not
have much of it left.



We went back and tested the pH of the condition that had given crystals,
the expected pH was 7.9 but we found it to be 6 – 6.5 using a pH indicator
strip. We believe the drop in pH is caused by oxidative degradation of the
PEG1000 resulting in the formation of carboxylic acid species.


We have contacted Jena Bioscience to try and get some of the old screen
stock but unfortunately they do not have any.


My question is does anyone out there happen to have any expired screen
stocks of this Pi-minimal condition (#57), ideally from the same lot (lot #
JB200013133), that they would be willing to send us.



Does anyone have any advice as to how to reproduce the condition? We’ve
considered bubbling oxygen through and heating the sample to accelerate the
oxidation process.



King Regards


Jonathan Bailey (PhD student)


Professor Martin Caffrey Lab MS&FB group Trinity College Dublin