Hi Dipankar,
I've produced several serine protease constructs in Ecoli and then refolded
them. I would investigate the following (some of which echoes previous
comments):
- try both urea and guanidine as your denaturant
- try refolding in stages (e.g. 8M urea to 2M to 100mM or equivalent)
-
Dear Tatiana,
Having worked also worked on various Fe(II) dependent hydroxylases, may I
suggest trying some other less redox sensitive divalent cations? My old lab
routinely used to screen hydroxylases with (to my knowledge) Co and Mn,
which essentially inhibited enzymatic turnover, so they were
Dear All,
Apologies in advance if this has already been posted, but I came across the
following short clips about crystallography recently and thought they may
be of interest.
http://www.theguardian.com/science/video/2013/nov/27/x-ray-crystallography-video
And just to add a little further to Mark's point, is there anyone here who
has at some stage received reviewer comments and NOT immediately spent the
next little while trying to deduce who each of the reviewers were?
(especially if they were negative...)
I would imagine most people have a pretty
Just to add to Herman's suggestions, if you are trying to crystallise a
protease then you could also try using the S195A variant rather than an
inhibitor. This would certainly be the case if you ever want to
co-crystallise in a substrate, as PPACK (or the like) would occupy the
active site cleft
Hi Juergen,
Your scheme as printed has two J's - so January and July are indistinguishable!
I would suggest the letters should instead be JFMAYULGSOND.
Happy apocalypse!
Tom
On 21 Dec 2012, at 01:52, Bosch, Juergen jubo...@jhsph.edu wrote:
May I introduce you to another fool proof way:
Dear Andrew,
I would suggest that Larousse may need to revisit their entry -
freeze-drying (in every context I have come across it) refers to
lyophilisation, which (i) specifically requires the formation of ice
crystals, and (ii) results in the removal of all of the resulting ice from
the sample.
Hi James,
You can get the PCT (Pre-Crystallisation Test) from one of the more
famous manufacturers of crystallography products - essentially you can
quickly screen your protein to get an idea if it is too dilute, too
concentrated, or somewhere in the middle. Of course, the results are
I'm further racking my brain to figure out a biological implication of this
behaviour, I thought something like plaque formation but I can't find
support in literature.
A good example of domain swaps involved in disease-associated
polymerisation is the polymerisation of serpins; while
Hi JK,
As mentioned, phosphate salts are the main disadvantage - you can get
round this by setting up two drops per well: one with your protein in
PBS, and the other with PBS only. That way you should be able to
quickly identify any hits that are due to salt, and which are likely
to be your
I would second that suggestion, I just had some bands sequenced to
identify internal cleavage sites within a protein and got very clear
sequence back.
Cheers
Tom
On Thu, Nov 25, 2010 at 11:20 AM, MARTYN SYMMONS
martainn_oshioma...@btinternet.com wrote:
I have had excellent service from Mike
Hi Hannes,
We have recently had a batch of phage contamination - one of the
symptoms was exactly as you described, when pelleting the cultures, a
smeary, fluffy pellet appeared. The other symptoms we had were that
during growth, the OD would rise up until ~0.5, then drop, a foam
would develop on
conformation, which is perfect for forming helices (and
doesn't involve a cofactor).
Best Regards,
Tom
--
Dr. Tom Murray-Rust
Department of Haematology
Cambridge Institute of Medical Research
Wellcome Trust/MRC Building, Hills Road
Cambridge CB2 0XY
On Thu, Mar 4, 2010 at 10:45 AM, Miles Pufall mpuf
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