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 I could see his point if I did have to.
As for the variance between samples, I don't know about >25%, but I
have observed multiple readings to have variance. I always take 3
readings on my nanodrop and then average them to deal with the
variance I see. I don't mind doing this because the instrument is so
fast, and I don't mind the cost at 6 ul of sample total.
The most variance I have seen is usually in spin columns, where I will
be doing a buffer exchange from a storage buffer (sometimes at ca. 20%
glycerol) into an assay or xstal buffer, and I have wondered to myself
if the variance I see could be due to incomplete mixing of a protein
sample betwen a viscous buffer at the bottom with the rest of the
buffer. I don't know how often other people find themselves in a
situation where they may be sampling their 2 ul from a
"micro-environment" that is not homogenous with the rest of the
sample, but with small volumes I think that be a problem. Food for
thought.
Filip, I would buy a nanodrop. It is much better than a
Bradford/cuvette and your students will love you for it. Cheers~
~Justin
Quoting aaleshin <aales...@burnham.org>:
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 concentrated samples, which we crystallographers
use, so Nanodrop is ideal instrument for our trade.
If the drop is within recommended volume like 1-2 ul for our model,
its size has a very small influence on the measurement.
Cuvettes will give a better accuracy provided you clean them properly.
I hated those times when I had to measure a concentration because of
a need to wash a cuvette. In a biological lab they are always dirty.
We switched to plastic disposable cuvettes for that reason...
Alex
On Jun 16, 2011, at 1:06 PM, Filip Van Petegem wrote:
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. Unless you want to
pre-mix 50ul and use a small quantity of that, which defeats the
purpose of miniaturization... It all depends on your applications
and sample availability, but if you want a very accurate
measurement, miniaturized volumes just won't get you the same
accuracy.
Cuvettes will give a better accuracy provided you clean them
properly. Just some water or EtOH is *not* enough...
Filip Van Petegem
On Thu, Jun 16, 2011 at 12:52 PM, aaleshin <aales...@burnham.org> wrote:
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...
Alex
On Jun 16, 2011, at 12:43 PM, Oganesyan, Vaheh wrote:
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 concentrations the accuracy and therefore
consistency may become 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] 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 absorption at 280nm under
denaturing conditions (6M Guanidine), and using calculated
extinction coefficients based on the composition of mostly
Tyrosine and Tryptophan residues (+ disulfide bonds). This method
is also old (Edelhoch, 1967), but very reliable.
One thing about the nanodrop: smaller volume = more evaporation.
On the demo we've had, I was so unimpressed with the precision
(>25% variability between two consecutive measurement) that we
didn't consider this instrument at all. So unless you just want a
'rough' estimate, I wouldn't recommend it at all. But most
respectable spectrophotometers will take cuvettes with 50ul
volumes - a big step up from 1ml volumes...
Filip Van Petegem
On Thu, Jun 16, 2011 at 12:15 PM, Arnon Lavie <la...@uic.edu> wrote:
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, 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, Bradford says 6 mg/ml,
Nanodrop 3 mg/ml. So while it is fun/easy to use the Nanodrop, I
am not sure how reliable are the measurements (your thoughts?).
So QUESTION 1: What are people's experience regarding the
correlation between Nanodrop and Bradford?
While researching the Nanodrop machine, I heard about the Implen
NanoPhotmeter Pearl.
So Question 2: Is the Pearl better/worse/same as the Nanodrop for
our purpose?
Thank you for helping us to advance to the next millennium, even
if it is nearly a dozen years late.
Arnon
--
***********************************************************
Arnon Lavie, Professor
Dept. of Biochemistry and Molecular Genetics
University of Illinois at Chicago
900 S. Ashland Ave.
Molecular Biology Research Building, Room 1108 (M/C 669)
Chicago, IL 60607
U.S.A.
Tel: (312) 355-5029
Fax: (312) 355-4535
E-mail: la...@uic.edu
http://www.uic.edu/labs/lavie/
***********************************************************
--
Filip Van Petegem, PhD
Assistant Professor
The University of British Columbia
Dept. of Biochemistry and Molecular Biology
2350 Health Sciences Mall - Rm 2.356
Vancouver, V6T 1Z3
phone: +1 604 827 4267
email: filip.vanpete...@gmail.com
http://crg.ubc.ca/VanPetegem/
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Filip Van Petegem, PhD
Assistant Professor
The University of British Columbia
Dept. of Biochemistry and Molecular Biology
2350 Health Sciences Mall - Rm 2.356
Vancouver, V6T 1Z3
phone: +1 604 827 4267
email: filip.vanpete...@gmail.com
http://crg.ubc.ca/VanPetegem/
