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
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