Hi everyone,
I am working with a membrane protein and normally measure my protein
concentration by diluting and then reading OD280 (1cm pathlength). I have
found this to be very consistent, if a bit time consuming.
At the syncatron, I had to use a Nanodrop for the first time to check some
SAXS dilutions I made on site and it was all over the map. We tested each
sample 8-10 times and saw large variations (i.e. 2.5-4.1mg/mL) on a single
sample. I used the Nanodrop to measure OD280 and calibrated it with the same
extinction coefficient I normally use.
Since I have dodecyl-maltoside in all of my solutions (which is the cause of
most of my problems), I was wondering if it was also the culprit here.
The detergent is probably lowering the surface tension of the drops.
It is possible that I was just in-experienced, but I had multiple people
from the facility helping me and we all had the same issue with my samples.
In addition, the Nanodrop would regularly complain about inconsistency
between the two readings and wouldn't accept readings/blanks at all.
Is it me or the membrane protein? And is there anything that I could do to
improve the readings the next time that I need to use it?
Thanks,
Chelsy
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Pascal
Egea
Sent: June-16-11 8:18 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old
Bradford.
I would like to add something about the NanoDrop versus NanoPearl,
I don't think that the path length is fixed on this instrument (the
NanoDrop) since if I recall well, the instruments sets the path length as it
scans through the droplet, hence the characteristic clicky noise that you
hear as the handle moves. This instrument requires recalibration every year
(or so according to the vendors, and this is of course not cheap) since
there is a moving part that can get out of alignment.
On the other hand, the NanoPear from Implen as a fixed geometry for the same
tiny amount of sample required. Thus you do not have to deal with moving
parts and recalibration.
We just bought a NanoPearl and I should also mention that this instrument
does both things: nano-drop size measurement like the NanoVue AND cuvette
size measurements (for OD600 or old school Bradford).
All the best,
On Thu, Jun 16, 2011 at 4:57 PM, Shaun Lott s.l...@auckland.ac.nz wrote:
Just to add my 2c worth...
The department here has a couple of nanodrops as a shared facility, one for
DNA/RNA and one for protein. It has been noticeable that over time people
has been getting decreased reliability of measurements on the latter machine
cf cuvette measurements, presumably due to the build-up of protein deposits
over time - so I would say that although it's easier to clean than a
cuvette, the nanodrop is not immune to the problem. The biggest issue I see
with the nanodrop is evaporation of sample. Even here in moist Auckland,
where RH is very often 80%+, taking a series of measurements with the
nanodrop over a period of just a minute or two shows increasing
concentration in the sample. So, for consistent results, one has to be
careful to measure quickly. It's probably fine for comparative measurements,
but as has been observed above, not great for super-accurate values for
biophysics, and I think rather operator dependent. But all our students are
super-careful, right? ;) Worth to note also that ProtParam calculates
extinction coefficients based on Gill von Hippel, (Gill, S.C. and von
Hippel, P.H. (1989) Calculation of protein extinction coefficients from
amino acid sequence data. Anal. Biochem. 182:319-326) who claim accuracy of
~5% for 'normal globular' proteins without extra chromophores. Whilst on
this subject, I would put in a plug for the good old BCA (aka Pierce) assay
for protein concentration. It's a little slow, but gets away from sequence
dependency somewhat as it is primarily dependent on the peptide backbone
rather than sidechains and works well in micro-titre plates etc. It is
certainly very superior to Bradford. (Smith, P.K., et al. (1985).
Measurement of protein using bicinchoninic acid. Anal. Biochem. 150 (1):
76-85. doi:10.1016/0003-2697(85)90442-7).
cheers
Shaun
--
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
314 Biomedical Sciences Research Building
office (310)-983-3515
lab (310)-983-3516
email pe...@mednet.ucla.edu
