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<mailto: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<tel:%28312%29%20355-5029>
Fax: (312) 355-4535<tel:%28312%29%20355-4535>
E-mail: la...@uic.edu<mailto: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<mailto:filip.vanpete...@gmail.com>
http://crg.ubc.ca/VanPetegem/
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