For accurate absorbance measurements we have been using a 10 ul
"tunnel" cuvette
for some time. It is still 1 cm pathlength, fits into standard specs,
one can manage even with 8 ul of a sample fed into the channel,
which can be rescued ~ 80%, no evaporation issues, the cuvette has to
be kept clean!
SET CCP4BB CONCEAL
Hi Chelsy, yes we had a lot of trouble with the nanoview during that run. Even
after going through the calibration procedure with the special fluid provided,
we still had inconsistent results even on standards. Finally, I carefully
cleaned the return light path of the instrument (a separate s
Hi everyone,
I am working with a membrane protein and normally measure my protein
concentration by diluting and then reading OD280 (1cm pathlength). I have
found this to be very consistent, if a bit time consuming.
At the syncatron, I had to use a Nanodrop for the first time to check some
S
On Jun 16, 2011, at 8:30 PM, Dima Klenchin wrote:
You recommended "determining extinction coefficients experimentally". How is
plugging number of specific residues into a formula constitute experimental
determination?
That is a deeply philosophical question!
Eventually, you'll be plugging in
The method is that by Edelhoch, mentioned a couple of times already in
this discussion.
You recommended "determining extinction coefficients experimentally". How
is plugging number of specific residues into a formula constitute
experimental determination?
It's also described in the paper by
I see, by the experimental determination of the extinction coefficient you mean
correction for the difference between unfolded (which can be computed
accurately) and folded proteins. Am I right?
Sorry for making this topic viral...
Alex
On Jun 16, 2011, at 5:06 PM, Machius, Mischa Christian wro
Again, the method described by Gill & von Hippel is based on statistical
averages. Mach et al. (Anal. Biochem. 1992, 200, 74) later revised these
values. Pace et al. (Protein Science, 1995, 4, 2411) again re-determined these
averages, so if anything, the values from Pace should be used. Pace als
I would like to add something about the NanoDrop versus NanoPearl,
I don't think that the path length is fixed on this instrument (the
NanoDrop) since if I recall well, the instruments sets the path length as it
scans through the droplet, hence the characteristic clicky noise that you
hear as the
The method is that by Edelhoch, mentioned a couple of times already in this
discussion. It's also described in the paper by Pace et al., the same paper
that the formula in ProtParam is from (ProtParam does not use the values
determined by Gill & von Hippel). Last time I looked into this, the con
Here is also a very effective method:
1Gill, S. & Hippel, P. v. Calculation of protein extinction coefficients
from amino acid sequence data. Analytical Biochemistry 182, 319-326,
(1989).
On Thu, Jun 16, 2011 at 5:56 PM, Filip Van Petegem <
filip.vanpete...@gmail.com> wrote:
> A convenient
Just to add my 2c worth...
The department here has a couple of nanodrops as a shared facility, one for
DNA/RNA and one for protein. It has been noticeable that over time people has
been getting decreased reliability of measurements on the latter machine cf
cuvette measurements, presumably due t
A convenient fast way is the earlier mentioned Edelhoch method, as described
in this paper which is referenced on the popular Protparam tool:
http://onlinelibrary.wiley.com/doi/10.1002/pro.5560041120/pdf
Filip
On Thu, Jun 16, 2011 at 4:45 PM, aaleshin wrote:
> Mischa,
> You intrigued me. What
Sorry for misprint, I meant evaporating water from a protein solution...
On Jun 16, 2011, at 4:45 PM, aaleshin wrote:
> Mischa,
> You intrigued me. What is the experimental technique for the Extinction
> Coefficient measurement (which requires knowledge of protein concentration)?
> Let me gue
Mischa,
You intrigued me. What is the experimental technique for the Extinction
Coefficient measurement (which requires knowledge of protein concentration)?
Let me guess, Bradford? Protein evaporation and weighing?
Alex
On Jun 16, 2011, at 4:22 PM, Machius, Mischa Christian wrote:
> With re
I think that the absolute value of protein concentration is not very important.
Some proteins get crystallized at 1 mg/ml, others at 50. What is important is
to be able to reproducibly estimate it from prep to prep. You probably want to
start at some reasonable value of about 10 mg/ml. If it in
With respect to the Edelhoch method and the ProtParam server, I would strongly
recommend determining extinction coefficients experimentally and not rely on
the ProtParam values. The reason is that the underlying extinction coefficients
in the formula used by ProtParam and referenced there are st
Totally support the statements below. We have had several proteins with A280
absorbance of 0.1 or less (at 1 mg/ml). You _have_ to use Bradford in the
Nanodrop or whatnot to measure the concentration.
Before purchasing the Nanodrop we used a Hellma TrayCell and a "normal" UV/Vis
instrument. Sim
Concerning "low resolution SAD phasing" I would like to add some information
here:
1. OASIS (version 2000) helped solving the unknown structure 1u9s in phasing
the 2.9A SAD data (
http://www.sciencemag.org/content/suppl/2004/09/30/306.5693.104.DC1/Krasilnikov.SOM.pdf
).
2. OASIS-2004 helped solvi
I have used the nanodrop and like it, although I have also seen some
variation, even sometimes a concentrating trend over ~30sec due to
evaporation I think. So, I always spot the protein then measure
instantly, and it is relatively consistent. But overall the
convenience is excellent, and worth the
> I have a 3.2A dataset for a protein-DNA complex. The protein is
> a homodimer, and the DNA is almost palindromic (except one base pair
> in the middle and two or three base pairs at both two ends). It is my
> first time solving structures, and unfortunately the resolution is
> low. No body
'Fine' for me means comparable to SEC-MALLS measurements and reproducible.
I use the E calculated from the sequence using the protparam server at Expasy.
David C. Briggs PhD
Father, Structural Biologist and Sceptic
University of Manchester
Hi Xun,
I'd find out what the NCS averaging correlation is between the molecules in the
asymmetric unit. If your correlation is high then tight NCS should help in
refinement. You could also take a look at the NCS averaged map in COOT. You can
also have ideal helices build using COOT or PHENIX (
Hi Xun,
I have a 3.2A dataset for a protein-DNA complex. The protein is a
> homodimer, and the DNA is almost palindromic (except one base pair in the
> middle and two or three base pairs at both two ends). It is my first time
> solving structures, and unfortunately the resolution is low. No
Hello Justin and others,
The volume comment I make is based on mixing prior to the experiment, e.g.
with Bradford reagent, Guanidine for the Edelhoch method, etc.
Any direct measurement of A280 of protein samples requires you to know the
extinction coefficient, which depends on the amount of Tyro
Arnon Lavie wrote:
~~~
We have been considering buying a Nanodrop machine (small volume, no
dilution needed, fast, easy).
However, while testing our samples using a colleague's machine, we have
gotten readings up to 100% different to our Bradford assay (all fully
purified proteins). For example,
On Thu, Jun 16, 2011 at 2:11 PM, Xun Lu wrote:
> I have a 3.2A dataset for a protein-DNA complex. The protein is a
> homodimer, and the DNA is almost palindromic (except one base pair in the
> middle and two or three base pairs at both two ends). It is my first time
> solving structures, an
Hi,
I have a 3.2A dataset for a protein-DNA complex. The protein is a
homodimer, and the DNA is almost palindromic (except one base pair in the
middle and two or three base pairs at both two ends). It is my first time
solving structures, and unfortunately the resolution is low. No body in ou
Hi Alex,
I read Filip's comment about volume not as a path length argument, but
about concentration uncertainty in mixing small volumes to dilute a
sample down before measuring it (?). I have never had to make a
dilution for my nanodrop (my proteins are usually not that
concentrated), but
Although the path length of NanoDrop is fixed. 1 ul may not form good liquid
column. One trick is to spot a little bit more sample, 2-4 ul, on Nanodrop,
specially for concentrated protein samples with glycerol and E. coli
culture. It eliminate the bubble problem.
To get good reading, a new drop n
Dear Arnon,
I have a Nanodrop2000, which reads from the post or a user supplied
cuvette. I have had NO complaints about using the Nanodrop for reading
protein concentration immediately prior to crystallization setup. When I
have observed differences in OD280 vs Bradford, it is usually due to one
o
I would add that there are some issues with air, you have to be careful with
nanodrop that the path is ok, and also if concentrations are low, <1 mg/ml for
instance, i am not sure one can trust it - compare 50 ul 1 cm path results with
nano at 0.5-1 mg/ml... i get inconsistency there.. its good
I'll give my backing to the Nanodrop as well.
I've used it in two different labs, for general yield checking use as
well as prior to ITC experiments, and haven't found there to be any
issues.
That said, I've also used cuvettes, and I find that one the whole,
cuvette-derived and nanodrop-derived m
Filip,
25% accuracy is observed only for very diluted (OD280< 0.1) or concentrated
samples. But those sample a rarely used for ITC or CD. The concentrated samples
require dilution but a regular spec does it too. Since the light passway is
very short in Nanodrop it is accurate with more concentra
On 2011-06-16 13:06, Filip Van Petegem wrote:
Even if evaporation is not an issue, one has to take pipetting errors into
account
when dealing with small volumes. The relative error on 1-2ul is a lot bigger
than on 50ul.
True, but the nanodrop works independent of volumes, since it has a
fix
We also have not experienced any problems with a Nanodrop 2000C.
No one in my touched the two boxes of Bradford and BCA kits that we
have, because we have been very happy with the Nanodrop.
Quyen
___
Quyen Hoang, Ph.D
Assistant Professor
Department of Biochemistry a
25% is not acceptable for ITC or CD experiments though...
I was just sharing our bad experience with a demo nanodrop we had. Even if
evaporation is not an issue, one has to take pipetting errors into account
when dealing with small volumes. The relative error on 1-2ul is a lot
bigger than on 50ul
I second Vaheh,
been using Nanodrops at three different locations and have been happy with them
plus reproducibility of results. If you have 50 mg/ml you'll need to dilute,
but we rarely get that high anyhow.
Additionally you safe time. If you had a cuvette based system, you should
always clea
Never had problems with evaporation (and this is in the relatively dry
climate of Denver, CO, especially in the winter when the relative
humidity is in the low 20%).
Using the Thermo Scientific Nanodrop 2000c.
We use it also as a prerequisite for ITC, which can be very sensitive
to proper
I also like our Nanodrop, but I do not recommend using it for Bradford
measurements.
The 25% accuracy mentioned by Flip is pretty good for biological samples.
Using 50 ul cuvette in a traditional spectrophotometer will not give this
accuracy because cleanness of the cuvette will be a big issue
I completely disagree with Filip's assessment. I've been using nanodrop nearly
5 years and never had inconsistency issues. If you work at reasonable speed (if
you put a drop there then lower the lever and click measure before you do
anything else) there will be no issues. At very high concentrat
Dear Arnon,
the Bradford method is not recommended for accurate measurements. The
readings are strongly dependent on the amino acid composition. A much
better method is using the absorption at 280nm under denaturing conditions
(6M Guanidine), and using calculated extinction coefficients based on
Dear fellow crystallographers - a question about spectrophotometers for
protein concentration determination.
We are so last millennium - using Bradford reagent/ 1 ml cuvette for
protein conc. determination.
We have been considering buying a Nanodrop machine (small volume, no
dilution needed,
UNIVERSITY OF CONNECTICUT (UConn)
POSTDOCTORAL POSITIONS, BURKHARD LAB
Crystallographic and biophysical studies of intermediate filaments
Postdoctoral positions (including a senior postdoctoral position) in
Intermediate Filament Structural Biology are available immediately for highly
motivated
I don't think it worked because I can still see your email address.
James
On Jun 16, 2011, at 10:27 AM, Peter Burkhard wrote:
>
> SET CCP4BB CONCEAL
>
>
> Peter Burkhard, PhD, Assoc. Prof. Nanobiotechnology
> The Institute of Materials Scie
SET CCP4BB CONCEAL
Peter Burkhard, PhD, Assoc. Prof. Nanobiotechnology
The Institute of Materials Science
University of Connecticut
97 North Eagleville Road
Storrs, CT 06269-3136, USA
Phone: +1 860 486 3830
Fax: +1 860 486 4745
E-mail:
Sean Seaver wrote:
Better yet, RbBr? and pH MES, MOPS, and HEPES with RbOH?
I haven't checked the price.
Including shipping in the US
RbBr: 50 g 99% pure is ~$150.
RbOH: 25g 99+% pure in 50 % water runs about ~$120.
It would be interesting to see how the substitution would influence the
crys
Hi,
On Thu, Jun 16, 2011 at 7:49 AM, Jan Dohnalek wrote:
>
> Modeling more UNKNOWN atoms might be the future for these cases?
>
one needs to specify chemical element type in 77-78 position, otherwise
these records are useless. Example:
http://www.rcsb.org/pdb/files/3GR4.pdb
HETATM15389 UNK U
>Better yet, RbBr? and pH MES, MOPS, and HEPES with RbOH?
>I haven't checked the price.
Including shipping in the US
RbBr: 50 g 99% pure is ~$150.
RbOH: 25g 99+% pure in 50 % water runs about ~$120.
It would be interesting to see how the substitution would influence the
crystallization or cryst
Maybe all those highly disordered bromides (and rubidium ions) would be
difficult to model and would push up the R factors? From the point of
view of SAD or MAD phasing, a large number of partially occupied sites
might be difficult to find. The highly disordered sites might also
create an anomalo
Yes! Although the Rb edge might be a little tricky to get to, but the
extra density would probably show up. Also, does Rb substitute well
for Na?
Rb:
Edge keV A
K15.19970.8157
I was thinking that perhaps the reason that solvent HAs were not used
historically is th
Better yet, RbBr? and pH MES, MOPS, and HEPES with RbOH?
I haven't checked the price.
Jacob Keller wrote:
Is there any reason why crystallographers have not routinely
substituted NaBr for NaCl in protein crystallization stocks, or even
pH'd their TRIS with HBr, if there is no NaCl? Wouldn't it m
Is there any reason why crystallographers have not routinely
substituted NaBr for NaCl in protein crystallization stocks, or even
pH'd their TRIS with HBr, if there is no NaCl? Wouldn't it make a lot
of sense, since there would always be a possibility for a Br-
derivative, and the price difference
We often fight these questions when "various ligands" bind to our proteins.
Generally, even if we know it's not water but an unidentified ligand
which cannot be properly modeled or we are not brave enough to place
it in the density we leave majority of the density uninterpreted but
DO model a few w
Tommi,
Since you ask, SHELX and other 'direct methods' run out of steam at about
1.2A and for many years very few unknown structures have been solved at
lower resolution than 1.2A. Recntly this picture was changed by the
introduction of ARCIMBOLDO, which has solved a number of structures ab
init
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