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 <> 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:
> ***********************************************************

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

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