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
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
Giulliana,
Alternatively to the previous excellent suggestions, you can add a few more
residues between your protein and TEV-cleavage site. For example, if you have
tag on the C-terminus you can add GSGS after the last residue: it should
improve cleavage without significant impact on
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)
On 05/02/2015 12:23 PM, Imre Berger wrote:
Dear Edward -
Would you be so kind and explain why you went ahead to post that comment
about Alex Rich on CCP4, in a thread which announced the sad news of his
passing away?
Yes- I realized after posting it that it was inappropriate. If there is any
