Dear Arnon,
I have a Nanodrop2000, which reads from the post or a user supplied
cuvette. I have had NO complaints about using the Nanodrop for reading
protein concentration immediately prior to crystallization setup. When I
have observed differences in OD280 vs Bradford, it is usually due to one
of the following:
1) The protein is deficient in the amino acid residues that provide the
280nm signal. This can be corrected with the extinction coefficient, and
can be programmed into the Nanodrop so that the readout is correct.
2) The protein is behaving badly in the Bradford assay (interference
with some component of the protein buffer)
3) The dilution used in the Bradford is contributing to large
concentration errors (and can be combined with 2, above)
The value of the Nanodrop, is that you get a OD280 that you can
reproduce prior to every experiment at a low cost of protein sample. The
N2000 can also do the Bradford or other assay on the post, or with a
cuvette if you really want to do it that way. When there have been no
mitigating factors, my Nanodrop OD280 readings have been within 5% of
the values I get from SEC-MALS, MassSpec or Bradford. I have no
experience with the Pearl, but I am very happy with my Nanodrop 2000.
Good luck with your choice!
Bryan
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-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of
Arnon Lavie
Sent: Thursday, June 16, 2011 3:16 PM
To: [log in to unmask]
Subject: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old
Bradford.
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: [log in to unmask]
http://www.uic.edu/labs/lavie/
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