''Signals in the region from 250-270 nm are attributable to phenylalanine
residues,
signals from 270-290 nm are attributable to tyrosine, and those from 280-300 nm
are attributable to tryptophan. Disulfide bonds give rise to broad weak
signals throughout the near-UV spectrum.
''
Dear all,
A question for the biochemistry-inclined folks in the bb; how do I
calculate protein concentration of chromatography fractions, starting from
Abs280 from the UV monitor? I know I could figure it out myself if I really
tried, but why bother when I have access to so many brilliant
Step1:
visit Protparam tool and CP your sequence, scroll down until you find the
extinction coefficient part. If OD280 is not close to 1 then make sure to take
that into account in your chromatogram say it is 0.7
step2: look at your mAU's
If your peak is 1000 mAU then you have 0.7 mg/ml in that
Hi Karel -
To add to what Jurgen said, a few points on the measurement of the
protein peak from the chromatogram.
- I usually approximate the peak as a triangle, so that the total peak
area is 1/2 height (absorbance maximum) x base (the number of ml in your
pool)
- If your peak is a
On 1/15/14, 9:34 AM, Matthew Franklin wrote:
- I usually approximate the peak as a triangle, so that the total peak
area is 1/2 height (absorbance maximum) x base (the number of ml in
your pool)
GE's UNICORN for AKTAs (and probably the new Biorad FPLC software)
integrates and tells you the
I am not sure, but probably path length not 1 cm in a detector cuvette .
You must refer to your HPLS system manual for this value
15.01.2014 19:09, Karel Chaz пишет:
Dear all,
A question for the biochemistry-inclined folks in the bb; how do I
calculate protein concentration of chromatography
Thanks for all the replies. And I would not call this laziness, rather
crowdsourcing
I should have added a few more details; it is easy to export from say
Unicorn to excel, a list of pairs of values, Abs280 vs elution times, with
which one can recreate the chromatogram. I wanted to use these
I don't think it possible for protein purification purpose. There would be no
linear correlation between A280 and the protein concentration.
For pure protein analysis, as for minor amount protein used, it is possible.
Acoot
From: Karel Chaz
If using Unicorn,
1)Open your chromatogram in Evaluation window.
2)Go to Operations- Pool
3)Choose which baseline estimation suits you, define the pools (numbered rulers
that appear under the chromatogram)
4)type in the path length (2 or 10 mm for UV-900 and 2 mm for UPC-900)
5)type in the mass
Hello message board,
My group has some crystals of an interesting protein to take to the synchrotron
in a couple of weeks. We won't be able to prepare and crystallise a SelMet
derivative during that time period, but we have loads of crystals sitting
around. The diffraction isn't great, we see
free cysteines? - pCMB
phosphate-binding? - tungstate
Best,
Matthias
-
Dr. Matthias Zebisch
Division of Structural Biology,
Wellcome Trust Centre for Human Genetics,
University of Oxford,
Roosevelt Drive,
Oxford OX3 7BN, UK
Phone (+44) 1865 287549;
Fax
You don't mention the condition, which is important (esp pH). Best pH values
are 6-8.
From personal experience: try K2PtCl4 and OsCl3.
Try ~5 mM for 30-60' (quick soak).
The advantage of osmium salts is that they give a nice color to your crystals
so you know something is binding.
HTH and GL,
There is quite a bit of literature on this, but my favorite paper is this:
http://www.ncbi.nlm.nih.gov/pubmed/18391402
Towards a rational approach for heavy-atom derivative screening in
protein crystallography.
Spoiler: The overall winner is ethyl mercury phosphate (Figure 7). Of
course,
More references to consider…
You asked about soaking times - here are two articles advocating quick soaking
at relatively high heavy atom concentration, which has worked well for us.
We've had good luck with thimerosal.
Acta Crystallogr D Biol Crystallogr. 2002 Jul;58(Pt 7):1099-103. Epub
And quick iodide soaks may be useful in the 200 mM range. See the sped-up
video:
http://www.youtube.com/watch?v=45Qc3jOPaKY
Quiz time: What wavelength would give iodide a similar signal to that of
selenium? Can one get a better signal than selenium by choosing a different
wavelength for
On Jan 15, 2014, at 10:27 AM, Jim Pflugrath wrote:
Quiz time: What wavelength would give iodide a similar signal to that of
selenium? Can one get a better signal than selenium by choosing a different
wavelength for data collection?
I'll bite,
At ~11,000eV Iodine has about 3.8
Hi
do not forget the clusters like Ta6Br12 or the lanthanides.
in case your interesting protein is a membrane protein there are some
choices that might work better than other
we have described it here.
http://www.ncbi.nlm.nih.gov/pubmed/16855303
This is not exclusive to membrane proteins at
***
Research Officer in Dynamic Structural Science at the Research Complex
at Harwell and the University of Bath (fixed term for 2 years)
***
Dear Rhys,
an important addendum to the magic triangle is the fact that it gives
you direct evidence whether or not the substructure search has succeeded
(by locating the triangle in the substructure solution) so that you can
carry on with phasing via density modification. At 3.5A resolution this
Keith's favourites over many years (shared with Gideon):
K2PtCl4
K2PtCN4
KAu(CN)2
Ethyl Mercury Thiosalicylate (gentle Hg reagent)
Try for about 2-12 hours at 1 mM initially. If crystals crack, reduce
concentration - at least they bind!
If these don't work, then finding a derivative is
Also, if you have a sample changer for easy screening, try 1 mM, 5 mM 20 mM of
each
for 1h, 5h, overnight, and screen all ~60 crystals for diffraction
overnight.Send those that survive
to the synchrotron.
K
On 15 Jan 2014, at 17:18, RHYS GRINTER wrote:
Hello message board,
My group has
Hi Rhys -
Don't forget to try sulfur-SAD, especially the multi-crystal version
published recently:
http://journals.iucr.org/d/issues/2013/07/00/ba5189/index.html
This seems well suited to your situation.
- Matt
On 1/15/14 12:18 PM, RHYS GRINTER wrote:
Hello message board,
My group has
Hi Ursula,
you will find answers here:
http://www.phenix-online.org/papers/he5476_reprint.pdf
Pavel
On Wed, Jan 15, 2014 at 1:38 PM, Ursula Schulze-Gahmen
uschulze-gah...@lbl.gov wrote:
I am submitting a structure to the PDB database. The SfCHECK summary
report provided by the PDB
Did anybody mention native gel electrophoresis to select suitable HA ions?
Worked for us really nicely in a situation were speed was essential.
Here's a reference: PMID 14646083
Klaus
===
Dr. Klaus Fütterer
Room 717,
Hello Rhys Grinter, hello to the ccp4bb community,
I don't necessarily want to advertise here one of the major research
topic of Eric Girard and late Richard Kahn lab (my previous lab ;-) ) but...
You could maybe try lanthanide complexes ?
They are composed by a chemical ligand, which can
Dear Rhys,
You may consider Xenon derivative, which could be prepared simply pressurizing
the protein crystals in a xenon chamber. It does not require any modification
of mother liquor. It just needs cryo-protectant where crystals are stable for
at least one to two mins. Higher Pressure (20
Dear all,
We all know that for purified protein, A260/A280=0.5, and for purified DNA,
A260/A280=1.8. Now my protein A260/A280=0.75, So I wonder if there is DNA
mixed with protein? Is there any way I can use to test if there is DNA?
Thanks~
You may be missing a trick by not using metals as crystallisation additives if
your best
diffraction is only 3.5 Angstroms. Uranium has to be my favourite heavy metal,
even if
I've solved more structures with Pt and Hg. It gave crystals diffracting to 1.2
A from a
protein that otherwise gave
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