Hi Everyone,
Thank you so much for your additional tips about various kits. My protein
is tagged and when I cleave off the tag (a step that needs reducing agent),
I will unfortunately have both detergent and reducing agent in my protein
buffer.
Nicolas, thanks for your word of caution. I can't th
Hi Everyone,
After several attempts to cleave the SUMO tag off my membrane protein under
various conditions (different reducing agents, enzyme-to-substrate ratios,
etc.) and after reading the manual and troubleshooting guide, I'm reaching
out to the ccp4bb community.
Experiment: I dialyze the mix
Dear Kay,
> Concerning usage of programs, everybody has his/her preferences, but what
> could be simpler than a 2-liner XDSCONV.INP like
> INPUT_FILE=XDS_ASCII.HKL
> OUTPUT_FILE=temp.hkl CCP4 ! or CCP4_F or CCP4_I or SHELX or CNS
> and then running XDSCONV by running "xdsconv"? At least there's
Hi Richard,
Do you require visible light only?
In any case, i remember a neat paper describing using Hoffman modulation, as
well as polarisation and phase contrast. Pure analog computing ;) What stroke
me then (and now) is that the standard microscopy kit people use does not use
the variety
Hi Derek
I strongly recommend comparing with pointless/aimless, *not* pointless/
scala (if you have the time!)
Scala is obsolete and was superseded by Aimless several years ago (I
think 2010, without checking...). I find that Aimless is not only much
faster than Scala, but also scales bett
Hi Raji,
Your membrane protein should be in micelles as a protein-detergent
complex. The detergent belt buries much of the protein surface area and can
make a lot of biochemistry experiments less efficient.
I've never worked with SUMO tags before, but if cleaving them is like
with oth
Dear all,
I would like to introduce XDSIT, a command-line/GUI/database tool for automated processing of single or multiple diffraction datasets based on processing by XDS.
http://michaelkrug.reussmedia.de/
Best wishes
Michael Krug
Hi Raji,
I have no experience with membrane proteins, but I have used SUMO tags
frequently. Unlike other proteases that cleave at a specific site
(thrombin, TEV, etc.), Ulp1 recognizes the tertiary structure of SUMO for
cleavage. So if only about 50% of your protein is cleaved, this may
indicate
You could also try TCEP as a reducing agent-strong and compatible with IMAC.
JPK
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Raji
Edayathumangalam
Sent: Sunday, February 16, 2014 10:58 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Trouble cleaving SUMO tag off of membr
By the way, I have an unrelated question. In the crystal structures
containing yeast SUMO (including Ulp1-SMT1 complex: 1EUV), the first ~19
residues are absent. I am curious whether people tried a SUMO tag with
these residues deleted. I am using the vector from invitrogen which seems
to have the f
Sorry for my typo, it is Ulp1-SMT3 complex...
On Sun, Feb 16, 2014 at 10:54 PM, Chen Zhao wrote:
> By the way, I have an unrelated question. In the crystal structures
> containing yeast SUMO (including Ulp1-SMT1 complex: 1EUV), the first ~19
> residues are absent. I am curious whether people tr
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