I also have to come in defense of the nanodrop here.
I have measured up to A280 = 98 and the curve is always reasonably
smooth, spikes normally mean that bubbles have formed. And "proper
cleaning" seems to be rubbing with a kimwipe three or four times
after each drop. If the last user does not clean after the last
measurement, then things will dry up in the pedestals and that can of
course be a problem. Also bubbles do not form if you are careful. I
always use 2uL, 1uL can be too little, and even with 20% detergent I
get a column and no bubbles nearly every time. You have to pipet
carefully and lower the lever slowly (think of a vinyl record here).
For tricky samples, use 2.2uL instead. A bubble might still form
occasionally, but looking at the spectrum will quickly tell you that
something went wrong.
Cheers,
Jose.
**************************************
Jose Antonio Cuesta-Seijo
Cancer Genomics and Proteomics
Ontario Cancer Institute, UHN
MaRS TMDT Room 4-902M
101 College Street
M5G 1L7 Toronto, ON, Canada
Phone: (416)581-7544
Fax: (416)581-7562
email: [EMAIL PROTECTED]
**************************************
On Dec 5, 2008, at 7:00 PM, wangsa tirta ismaya wrote:
Dear all,
Thanks a lot for raising the issue with the not reproducibility of
protein measurement with Nanodrop. We use the instrument as a
workhorse in the lab. Indeed, recently I observed that the protein
concentration suggested by Nanodrop is sometimes differ to the
usual colorimetric measurement (Bradford method, measured with our
Pharmacia's Ultrospec 2000 spectrometer). Since the cell in
Nanodrop is very small, could it be due to the homogeneity of the
sample in the cell? Also what I have observed, we have to be sure
that the cell is cleaned properly before use. Another thing is, at
high protein concentration I obtained noisy absorption curve at the
peak (like a seismograf ....) thus the protein concentration
measured is doubtful, I have to dilute the sample to have good
curve (thank God it requires only 2 mikroliter for a measurement).
Well, I think nanodrop is a good, fast, and powerful instrument,
however it would be better if we established a reference of our
daily practice to a normal spectrometer measurement.
cheers,
Wangsa
2008/12/5 Martin Hallberg <[EMAIL PROTECTED]>
Which brings us back to the Hellma "TrayCell" solution where you
can, from the same spectrometer, have both the cuvette option and
the quickness of the NanoDrop/NanoVue system.
Anyone that can comment on the performance of the TrayCell from
Hellma?
Cheers,
Martin
On Dec 5, 2008, at 9:06 AM, Gregor Witte wrote:
Agree!
I think for crystallographic use the nanodrop is perfectly okay to
see if the protein is 5mg/ml or 30mg/ml. But in fact I also do not
trust our instrument if it comes to more important issues like
preparing solutions for titrations or assays. And due to the small
pathlength I do not trust absorptions of small concentrated samples
at all. I always prefer a "real" 2-beam spectrophotometer
(monochromators) with a quarz-cuvette and a nice pathlength. Of
course, you cannot reliably measure solutions exceeding Abs 1 or
maybe 1.5 OD in a spectrophotometer with 1cm pathlength.
There's also one quite strange thing about the nanodrop – they sell
the "calibration check solution" (which is some kind of yellow
chromate-solution with known absorption), then you check your
nanodrop with it and maybe find out that it's off to some certain
extend: But then you're stuck(!), because you cannot calibrate it
on your own. I guess it would be quite easy to integrate a
calibration-option into the software, but at the moment the
instrument tells you "calibration failure" and you have to call the
service guys who then carry it home and calibrate it by turning of
the two small screws at the top of it and then glue them with
locktite.
Anyway, at least for our mid to high concentrated samples the
nanodrop is not showing large fluctuations so we are happy with it.
But everyone using a nanodrop should check it from time to time –
as I found out that ours was off more than 20% at one day - which
raised some trouble of course…
Cheers,
Gregor
Von: CCP4 bulletin board [mailto:[EMAIL PROTECTED] Im Auftrag
von Filip Van Petegem
Gesendet: Donnerstag, 4. Dezember 2008 22:20
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] suggestions for UV spectrometer
I want to add I absotely hate the nanodrop. We've had a demo for
it, and found the readouts to be very unreliable. Fluctuations of
20% and more. Just leaving the same drop in and measuring the
sample multiple times gives different values (going in both
directions, so not only due to evaportations). Sure, it's easy and
fast, and maybe good to have a rough idea about your protein
concentration, but I would never want to use it for exact
measurements such as needed for e.g. a CD or an ITC instrument.
I've heard other labs in our department have similar issues. We've
also had a demo for the Nanovue from GE Healthcare: same issues -
very large fluctuations from one sample to another. I suppose this
is simply an inherent problem with small volumes...
Cheers
Filip Van Petegm
On Thu, Dec 4, 2008 at 12:48 PM, Patrick Loll <[EMAIL PROTECTED]>
wrote:
At the risk of dragging this discussion even further afield from
crystallography:
How can you get realistic numbers for concentrated solutions using
the Nanodrop? I understand that the instrument reduces absorbance
by using a very short path length. However, I thought that in order
for the Beer-Lambert formalism to be applicable, the solution needs
to be sufficiently dilute so that the chance of molecules
"shadowing" one another is negligible. Isn't this condition
violated for concentrated solutions (even with short path lengths)?
Pat
On 4 Dec 2008, at 1:27 PM, Michael Giffin wrote:
We also like the Nanodrop...
----------------------------------------------------------------------
-----------------
Patrick J. Loll, Ph. D.
Professor of Biochemistry & Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA 19102-1192 USA
(215) 762-7706
[EMAIL PROTECTED]
--
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: [EMAIL PROTECTED]
http://crg.ubc.ca/VanPetegem/
.
B. Martin Hallberg, PhD
Assistant professor
Department of Cell and Molecular Biology
Medical Nobel Institute
Karolinska Institutet
Von Eulersv. 1
SE-171 77 Stockholm
Sweden
Fax: +46-8-339380
--
Wangsa Tirta Ismaya
=====================================
Josef-Israelstraat 66, 9718 GN Groningen
The Netherlands
**************************************
Jose Antonio Cuesta-Seijo
Cancer Genomics and Proteomics
Ontario Cancer Institute, UHN
MaRS TMDT Room 4-902M
101 College Street
M5G 1L7 Toronto, ON, Canada
Phone: (416)581-7544
Fax: (416)581-7562
email: [EMAIL PROTECTED]
**************************************
