To add to this discussion:
Many of the comments to my question that started this thread do not
sufficiently differentiate between accuracy and precision.
While we all want an assay that is internally consistent (i.e., high
precision), we do care a lot about accuracy ("the degree of closeness of
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!
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
time that I need to use it?
Thanks,
Chelsy
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Pascal
Egea
Sent: June-16-11 8:18 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old
Bradford.
I would like to add some
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
bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Machius,
Mischa Christian
Sent: Thursday, June 16, 2011 7:23 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old
Bradford.
With respect to the Edelhoch method and the ProtParam server, I would stro
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
5% 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...
>>>>
>>>> Alex
>>>>
&
'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
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 concentrations
>>>> the accuracy and therefore consistency may become lower. Conce
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,
lower. Concentrations between 5 and 10
mg/ml should be fine. The instrument is pricey though.
Vaheh
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf
Of Filip Van Petegem
Sent: Thursday, June 16, 2011 3:34 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Nanodro
ed in publications will help the research community.
Cheers,
Chun
-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bjørn
Panyella Pedersen
Sent: Thursday, June 16, 2011 1:19 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Nanodrop versus Nanophotomter
board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Arnon Lavie
Sent: Thursday, June 16, 2011 3:16 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old
Bradford.
Dear fellow crystallographers - a question about spectrophotometers for
protein concentration
e lower.
>>> Concentrations between 5 and 10 mg/ml should be fine. The instrument is
>>> pricey though.
>>>
>>> Vaheh
>>>
>>>
>>>
>>> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Filip
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
>
>>
>>
>> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Filip
>> Van Petegem
>> Sent: Thursday, June 16, 2011 3:34 PM
>> To: CCP4BB@JISCMAIL.AC.UK
>> Subject: Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus go
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
:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of
> *Filip
> Van Petegem
> *Sent:* Thursday, June 16, 2011 3:34 PM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good
> old Bradford.
>
>
> Dear Arnon,
>
>
> the Bradford
AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old
Bradford.
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
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
gt;
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Filip
> Van Petegem
> Sent: Thursday, June 16, 2011 3:34 PM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old
> Bradford.
>
> Dear Arnon,
>
> t
tegem
Sent: Thursday, June 16, 2011 3:34 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old
Bradford.
Dear Arnon,
the Bradford method is not recommended for accurate measurements. The readings
are strongly dependent on the amino acid compositio
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,
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