Hi Folks,
The paper Phil recommended looks very nice. Below is a summary when
last time I asked people about this.
Regards,
Weikai
-------- Original Message --------
Subject: [ccp4bb] Summary: measure detergent concentration
Date: Wed, 28 Oct 2009 16:49:32 -0400
From: wei...@crystal.harvard.edu
To: CCP4BB@JISCMAIL.AC.UK
Dear All:
Thanks a lot for all the responses. My question was about measuring
the
detergent concentration after concentrating a membrane protein sample.
Here is the summary.
1. The simplest way to control the detergent concentration is to use a
higher cut-off concentrator if you protein plus detergent micelle is
large
enough. Michael Matho: “a 50kDa cutoff withheld a lot of detergent
during
concentration process and consequently your final concentration might
increase significantly. For example we started with 0.25% DES and
noticed
increases of above 1%. This did not happen when using a 100kDa cutoff,
and
DES concentration remain pretty much constant.” It is easy enough to
test
the “maximal cutoff you can use w/o loosing your membrane protein in
the
flow through”.
2. Patrick Loll, Edward A. Berry and John K. Lee suggested TLC, which
seems to have the least requirement for equipments -- “silica gel TLC
plates and a chromatography jar”.
Patrick: “We've done this as an exercise in NSLS Membrane Protein
Crystallization workshop for a few years, and it works like a charm.
You
can stain in a warm iodine chamber and visualize by scanning the TLC
plate
on a garden variety scanner (we use an inexpensive Canon LIDE that
probably cost less than USD 60 five years ago). We quantify the spot
intensity with NIH Image or equivalent, and get lovely linearity down
to
the CMC, spotting only 1 uL of sample--so we haven't seen any need to
concentrate.
Edward: “spotting on a TLC plate and running beside standard amounts of
the same detergent. From intensity/size of the detergent spot after
developing you can bracket the detergent concentration. (And by the way
they found that detergents are concentrated by ultrafiltration). To
increase sensitivity, speedvac a volume too large to spot on the plate,
dissolve the residue in MeOH.”
A strategy for identification and quantification of detergents
frequently
used in the purification of membrane proteins. Laura R. Eriks, June A.
Mayor, and Ronald S. Kaplan. Analytical Biochemistry 323 (2003) 234–241
3. For sugar-based detergents (maltosides and glucosides), one can use
some traditional chemistry to measure the sugar.
Bert Van Den Berg: “do a fehling-type assay”
Zhenfeng Liu: ”phenol-sulfuric acid reaction for quantification of
sugars.” Biochemistry, vol 36, no. 19, 1997, p. 5887
Hari Jayaram: “sulfuric acid and phenol followed by Absorption
measurement; using a standard curve against the same detergent ”.
Anal
Biochem. 2005 Jan 1;336(1):117-24. A colorimetric determination for
glycosidic and bile salt-based detergents: applications in membrane
protein research. Urbani A, Warne T.
4. Christopher Law: Use surface tension properties and look at the drop
shape (measure contract angle). “A small droplet of the detergent
solution
is deposited on a piece of Parafilm M and side views are recorded by
two
orthogonally arranged TV cameras.”
A Novel Method for Detergent Concentration Determination. Biophys J.
2006
January 1; 90(1): 310–317. Thomas C. Kaufmann, Andreas Engel, and
Hervé-W.
Rémigy.
5. Ezra Peisach: by Refractive index. “Refractive index measurements
were
performed using an OPTILAB DSP instrument (Wyatt Technology) with a P10
cell.”
Refractive index-based determination of detergent concentration and its
application to the study of membrane proteins Pavel Strop and Axel T.
Brunger. Protein Sci. 2005 August; 14(8): 2207–2211.
6. Michael Matho: NMR is the most accurate method
“using a high detergent concentration stock solution you can assign
resonance peaks to your detergent molecule bonds. Then you can set up a
standard curve using different known detergent concentrations (for
example
from 10% down to 0.1%) by calculating the surface of your peak(s) which
is
directly related to your detergent concentration. Each time you need to
know the concentration of a new sample, you just need to record the
peaks,
and use the three-click rule to deduct the unknown value.”
7. David Veesler and Kornelius Zeth suggested ATR-FTIR (Fourier
transform
infrared spectroscopy). “very accurate, fast (10min) and requires as
low
as 10uL of protein sample.”
PVeesler, D. et al. Production and biophysical characterization of the
CorA transporter from M. mazei. Analytical Biochem. (2009). 388
:115-121.
Regards,
Weikai
