optimal cleavage conditions,
but it’s a very simple experiment to do.
Dave
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Giulliana
Rangel
Sent: 02 May 2015 18:57
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Tev cleavage
Dear all,
I'd like some help about my pr
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Giulliana
Rangel
Sent: Saturday, May 2, 2015 10:57 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Tev cleavage
Dear all,
I'd like some help about my protein cause I've a lot of problems in cleavage
moment. In this
Hey Giulliana,
does your protein buffer contain high imidazole concentrations ( > 150 mM)? If
so you should try to exchange the buffer before cleavage, since the TEV
protease tends to precipitate at higher imidazole concentrations.
Schara
> Am 02.05.2015 um 19:56 schrieb Giulliana Rangel :
>
Hi,
I have used tev protease for tag cleavage during dialysis. In my case
always tev got precipitated not my protein. And cleavage was always
complete. I havE checked on the sds - page as wel.
On 2 May 2015 23:27, "Giulliana Rangel" wrote:
> Dear all,
>
> I'd like some help about my protein cause
These are the usual culprits
My buffers for cleavage and an on-column digestion
worked good. (see below)
Also most likely your TEV source (do not go cheap) enzyme is inactive
(gone bad). Get a clone for TEV and make your own TEV in the lab. It save
you a ton of money
.
10 mM Tris-HCl (pH 8.0)
15
crystallization.
Vitali
From: CCP4 bulletin board on behalf of Giulliana Rangel
Sent: Saturday, May 2, 2015 12:56 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Tev cleavage
Dear all,
I'd like some help about my protein cause I've a lot of problems i
Hey,
I always had very good results with cleaving with TEV at 4C overnight
(30min are to short at this temperature). Alternatively, I did the TEV
cleavage at 16-23C for 1-3 h. 37C might just be to warm for your protein...
Since some time I switched to the PreScission system instead of using
TEV
Dear all,
I'd like some help about my protein cause I've a lot of problems in
cleavage moment. In this step after aproximadately 30 minutes (37C) occur
precipitation almost 50% .
I tried control it:
- Protein diluition (no results)
- Cleavage 4C ( no cleavage)
-Modifying buffers: add 10% glycerol
Hi Anita,
so you tested your crystals inhouse, any idea how they do at the synchrotron ?
Still no diffraction ?
Since it's a hexamer I would expect the His-tag to be not so important and
would rather rescreen with seeding first to see if any other conditions might
result in diffracting crystals
I'd say since you obtained crystals with your tag it is not a disturbing factor
and either disordered or making contacts. So removing the tag you might end up
not getting crystals in the worst case. Now to the question why they don't
diffract. Did you test the old fashioned way at RT in capillar
e on the
> right hand side of the cleavage site. Adding one or two amino acids after
> the current cleavage site may help.
>
> Zhijie
>
> From: anita p
> Sent: Friday, April 08, 2011 5:10 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Tev Cleavage issue !!
> Thanks e
a p
Sent: Friday, April 08, 2011 5:10 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Tev Cleavage issue !!
Thanks everyone for your suggestions !
Artem has pointed out that low diffraction of the crystal might be because of
other problems .. If you could highlight a bit more on this issue
Thanks everyone for your suggestions !
Artem has pointed out that low diffraction of the crystal might be because
of other problems .. If you could* highlight a bit more on this issue it
would be helpful for me.*
I have tried to seperate the cleaved, uncleaved and TEV over MonoQ column
but there w
Dear Anita,
Sometimes the protein of interest has a relatively strong inherent binding
affinity to the IMAC
column. Have you tried to bind the cleavage reaction to an IMAC column
and then elute using a shallow imidazole gradient?
In fact, Porath developed IMAC chromatography as a tool for prote
Hi Anita,
We have had success setting up drops with TEV present. We simply added TEV at a
50:1 molar ratio and then set up the drops a couple of hours later. We went
from having twinned crystals at 3A to untwinned at 2A, the crystal form also
changed from orthorhombic to monoclinic, all in the
Hey Anita,
I would like to add to Artem's comment that you can also try and cleave the
protein at 30c for 2hr and then continue the cleavage overnight at 4c (you
should check and see that your protein can withstand 30c incubation for 2hr,
of course).
In regard to your non-diffracting crystals - you
For starters, you could re-clone the protein with e.g. just a His tag or
move the tag to another end, or put some distance between the end of TEV
site and the protein; or perhaps use no tag at all -- or a different one?
Is it possible that the tag is messing you up - yes. Is it 'probable' - I
can'
Hi Crystallographers,
I am working of 23 Kda protein with a Nterminal His tag and a TEV cleavage
site.
I am getting crystals with the his tag and tev site intact, but they dont
diffract.
*Is it probable that they dont diffract because of the extra his tag and
the tev site?*
I am trying to get
Hi Matthew,
By now, you have received many posts telling you both how efficient and
inefficient TEVp is. You might be confused. This seeming contradiction
can be explained by a few events, among many others: Inaccessibility of
cleavage site, absence of reducing agents, and presence of deterge
Ok, to sum up for the board, a good reference for this problem is at:
http://mcl1.ncifcrf.gov/waugh_tech/faq/tev.pdf
Thanks to everyone who responded.
Matthew
On Mon, May 24, 2010 at 9:27 AM, Matthew Merski <
mer...@blur.compbio.ucsf.edu> wrote:
> Hello all,
>
> I am working with a protein
Hi Matthew,
TEV protease is very robust. I normally digest with 1:100 ratio
according to the OD280. I normally digest at 4C for overnight around 16-18
hours. Make sure your tev protease site are not inaccessible and buried
inside.
best
Xiaohu
On Mon, May 24, 2010 at 12:27 PM, Matthew Merski <
_
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Matthew Merski
Sent: Monday, May 24, 2010 9:28 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] TEV cleavage problems
Hello all,
I am working with a protein that is expressed as with an N-terminal domain
that is normally
Hello all,
I am working with a protein that is expressed as with an N-terminal domain
that is normally cleaved for activation of the protein (and
crystallization). For in vitro reasons I've needed to switch the normal site
to a TEV site. However, even though the TEV site is in the same place as t
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