I got several comments on/off board for this. I have checked by Western
blotting if any expression was occurring. I did not see any expression in
either the inclusion body or soluble fraction.
It appears that I have solved the problem;
Several papers suggest that the 10-30 bases after the start
Dear Bonsor!
You have very good suggestions from Amit with multiple solutions you could
try. In addition, I just want to add few more experiences facing with low
expression yield and/or no expression:
1. For protein with high number of predicted disulfide bonds, you may want
to try Origami strain
Hi,
Well, sometimes proteins don't express even though they are from the same
family and their "cousin" behaved very well.
I found the followings helpful:
1. forget about the concept "this special strain *should* work because it is a
Rosseta/ pLysS etc." Sometimes it's a long mRNA, sometimes a r
We have been expressing a family of human proteins as inclusion bodies in E.
coli, which we can be refolded and crystallized. However, three members show no
expression either as inclusion bodies or as soluble proteins.
The genes were ligated into the pE21d vector with either a His-tag or no
Hi
ding to nucleic acids and then
slowly coming off as NA get hydrolyzed or physically broken down.
Artem
_
From: megha goyal [mailto:mgbio...@gmail.com]
Sent: Friday, October 30, 2009 6:32 AM
To: Artem Evdokimov
Cc: CCP4BB@jiscmail.ac.uk
Subject: Re: [ccp4bb] Inclusion bodies centrif
gt; goyal
> *Sent:* Tuesday, October 27, 2009 10:21 PM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] Inclusion bodies centrifugation
>
>
>
>
>
> Dear All,
>
>
>
> Our protein is expressed as inclusion bodies and I want to separate
> inclusion bodies from
agree with Michael, use low concentration of detergent. I normally resuspend
the cell in buffer and sonicate, spin down at 6000 g., resuspend again with
buffer containing small amount of detergent and resonicate, spin down again
at 6000 g. finally was with buffer again (resuspend and spin down).
B
As Artem and Ezra have already mentioned the basic aspects of
isolating IBs, I should point out that most people wash the pellet of
the first low-speed centrifugation 2-3 more times. While Ezra
suggests using increasing amounts of urea, you may have to tailor the
composition of the wash bu
Artem has already responded - but I believe you will pull down the
cellular debris with the IB.
In my prior experience, post centrifugation, you can wash the IB/debris
with increasing amounts of urea in you solubilization - and you may find
that the other cellular debris stays in solution befo
In other terms, no worries. You have the technology.
Artem
_
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of megha
goyal
Sent: Tuesday, October 27, 2009 10:21 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Inclusion bodies centrifugation
Dear All,
Our
Dear All,
Our protein is expressed as inclusion bodies and I want to separate
inclusion bodies from E.coli from the cellular debris after* *lysis of the
cells by sonication.
Can I do this by normal centrifugation? and if yes, at what speed?
Our centrifuge has maximum speed of 14000 rpm. Can we d
You can coexpress the enzyme with chaperone (e.g GroEL and GroES)very
often this is very successful. (There are bunches of ways though...)
Best wishes,
Arundhati
2008/6/23 yangliuqing <[EMAIL PROTECTED]>:
> Hello,everyone!
> Sorry to bother you!
> Now I have an enzyme cloned in p
Hello,everyone!
Sorry to bother you!
Now I have an enzyme cloned in pQE2 ,expressed in E.Coli M15 at room
temperature,but there are a lot of inclusion bodies,perhaps 90%,it is a big
trouble because I need to get much enzyme.
Is there anyone knows how to solve?
Thank you very
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