"Richard Buick" <Richard.Buick at btinternet.com> wrote:
:Hello
:I've been expressing His-tagged proteins in E. coli Origami cells, about 10
:different proteins in all. I was using a His-probe for detection and was
:getting only about half of the proteins expressed. Until I turned to
:specific antibodies for detection eg, anti-prolactin and suddenly all
:proteins were detected. All had been sequenced, the His-tag is 5` to the
:protein sequence. Does anyone know why some are detected and others not by
:the His-probe? This is a real problem because I had planned on using the
:His-tag for purification.
What anti His tag do you use? Some of such Ab is rather
insensitive.
Since you are obviously getting incredibly small amounts
of protein expressed (well overexpressed protein in
E.coli should be a major band identifyable by Coomassie,
no need for Ab), my guess would be that you don't have enough
sensitivity and some proteins are expressed at levels below
your detection background.
Every protein behaves differently in E.coli. You can't possibly
expect 10 proteins to be expressed well without careful optimization
for each of them separately (some will, many won't). The problem
is particularly severe when a gene product is toxic - and again,
there is no way to predict what will and how will be toxic for
E.coli.
Alternative that's been mentioned that you are getting
translation initiation beyond 6His point is IMHO very rare
thing (but certainly possible).
- Dima
