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 protease. This enzyme works better in my hands than the TEV protease.
If you need a really fast and efficient cleavage (15min / 4C), you can
use a HIS-SUMO tag which can be cleaved off using ULP1. This is the most
efficient cleavage in short time that I know.
Good luck!
Karsten
Am 02.05.2015 um 10:56 schrieb Giulliana Rangel:
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 and 5% glucose (no crystallization)
- Add salt (1M - no results)
- Add serial tev (500ul in the first time and more 500ul in second
time- 37C) total precipitation
- Crystallization with 7 histag ( poor crystallization, no diffraction)
Now I need to produce this protein with semet that became the protein
more hidrofobic, probably.
So, If anyone could help me...
Thanks in advance
Giulliana Rangel
--
Karsten Thierbach, Dr. rer. nat.
California Institute of Technology
Division of Chemistry & Chemical Engineering
Hoelz laboratory
1200 E. California Blvd., M/C 147-75
Pasadena, CA 91125, U.S.A.
