Hi,
Can anyone pls help me .....I have a mammalian protein domain cloned in pET28a vector. I have checked its expression in BL21DE3,Rosetta,TuNERpLysS....the protein goes to pellet at all IPTG conc and even at lower temperature-16oC. i have checked with western too-Anti-His antibody. i can not purify the protein from pellet using 8M Urea because I need the protein for structural studies...dont want to refold it again.
What i shud do?
0
1 reply to this topic
#2
KAUSHIK THAKKAR
-
- Active Members
-
- 24 posts
Enthusiast
0
Neutral
Neutral
Posted 29 December 2009 - 02:41 AM
Hello,
I do not know whether you do sonication or not?
If not you should do sonication, to lyse the cells so that protein if present in inclusion bodies would be released..!!
We generally, use about 30-40% amplitude, for 10 sec with interval of 20 sec, with 5 cycles at 4 degrees.
This should increase your yield.
Also, before sonication you can add about 0.1 volume of 10 mg per ml lyzozyme, which would further enhance cell rupture, obviously you would be adding a protease inhibitor.
regards,
Kaushik
I do not know whether you do sonication or not?
If not you should do sonication, to lyse the cells so that protein if present in inclusion bodies would be released..!!
We generally, use about 30-40% amplitude, for 10 sec with interval of 20 sec, with 5 cycles at 4 degrees.
This should increase your yield.
Also, before sonication you can add about 0.1 volume of 10 mg per ml lyzozyme, which would further enhance cell rupture, obviously you would be adding a protease inhibitor.
regards,
Kaushik
HMG, on Dec 8 2009, 03:24 PM, said:
Hi,
Can anyone pls help me .....I have a mammalian protein domain cloned in pET28a vector. I have checked its expression in BL21DE3,Rosetta,TuNERpLysS....the protein goes to pellet at all IPTG conc and even at lower temperature-16oC. i have checked with western too-Anti-His antibody. i can not purify the protein from pellet using 8M Urea because I need the protein for structural studies...dont want to refold it again.
What i shud do?
Can anyone pls help me .....I have a mammalian protein domain cloned in pET28a vector. I have checked its expression in BL21DE3,Rosetta,TuNERpLysS....the protein goes to pellet at all IPTG conc and even at lower temperature-16oC. i have checked with western too-Anti-His antibody. i can not purify the protein from pellet using 8M Urea because I need the protein for structural studies...dont want to refold it again.
What i shud do?
