Dear Rana,
I think you need to clear up some confusion about this experiment. MBP fusions
suffer from a number of drawbacks depending on what you are doing. First, did
you use the MPB domain to purify the fusion protein (with an amylose column)?
If so, you also purified native MBP from the
Dear CCP4
I am having a problem with cleaving my fusion protein and I would be
grateful if you advice me regarding this situation, I have an MBP-DHBx fusion
protein and I am trying to cleave it using TEV protease, I have tried different
ratios and different temperatures with different
You do not mention what buffer you are trying to do your cleavage in. You need
a reducing agent for TEV to work (e.g. reduced gluthionine, DTT,
mercaptoethanol). EDTA (0.5-1mM) is recommended as TEV is a cysteine protease
and the presence of divalent metal ions will/eventually inhibit TEV. TEV
Since you're seeing the MBP band, it sounds as if you're getting some cleavage.
If there were no cleave you would only see the fusion and TEV bands on the gel.
It may be that your protein is not stable/soluble without the MBP fusion.
Different buffer conditions may help if your protein is
suggest that its
coming from fusion protein. I feel cleavage site may not be accessible for the
protease. Might be adding few more residues might help.
Good luckSDY
Date: Sun, 4 Nov 2012 07:24:42 -0800
From: rna19792...@yahoo.com
Subject: [ccp4bb] protein cleavage
To: CCP4BB@JISCMAIL.AC.UK
Dear All
Thank you for all your replies, the buffer for the TEV protease that I have
used contains 50mM Tris-HCl, 150mM NaCl, 1mM EDTA, and 1mM DTT at PH= 8.0. I
have tried using this buffer without NaCl but the TEV protease precipitates
when dialyzing over night, as for using glutathione
@Cynthia,
On Nov 4, 2012, at 10:58 AM, Cynthia Kinsland wrote:
Since you're seeing the MBP band, it sounds as if you're getting some cleavage.
If there were no cleave you would only see the fusion and TEV bands on the gel.
I think that is a wrong assumption.
He did not specify if he sees the
I assumed, perhaps wrongly, that the fusion was homogeneous prior to cleavage
as the existence of the MBP band post-cleavage would not have been noteworthy
if it had also been there pre-cleavage.
A time course or at least a time zero sample is always a good idea.
Truncation products prior to
We once had a more-or-less MBP-sized fragment before cleavage, but
this turned to be a spontaneous mutation. This expression experiment
had been started from a glycerol stock with an unknown number of
growth cycles prior to expression.
Starting from a fresh transformation with the purified
Do you see the same MBP band (or corresponding to the same MW, in case it
isn't MBP) before cleavage, at the same total concentration of protein? If
not, your protein could be crashing out. Even so, if you use SDS and heat
up the sample, I am surprised that your protein just disappeared. TEV
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