Weikai,
We did it using NMR but you asked for a simple way so I guess I'm out of topic.
Anyway, since I believe it is the most accurate method, here it is: 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.
As a colleague answered you earlier, we noticed that 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%. Of course this will depend on the
concentration factor.
This did not happen when using a 100kDa cutoff, and DES concentration remain
pretty much constant.
Now, it will depend on your system: what detergent you are using, since micel
size and CMC are obviously the critical parameters here -- but also what
maximal cutoff you can use w/o loosing your membrane protein in the flow
through...
Good luck,
Michael
----- Original Message -----
From: Patrick Loll
To: CCP4BB@jiscmail.ac.uk
Sent: Friday, October 23, 2009 1:12 PM
Subject: Re: [ccp4bb] measure detergent concentration
I'll second this. 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.
On 23 Oct 2009, at 3:41 PM, Edward A. Berry wrote:
Only easy if you happen to have silica gel TLC plates and
a chromatography jar lying around, perhaps from some
phospholipid analysis:
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
This paper recommends 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 Me0H.
Ed
wei...@crystal.harvard.edu wrote:
Hi Folks:
After concentrating a membrane protein, is there a (easy) way of measuring
the detergent concentration in the sample?
Regards,
Weikai
---------------------------------------------------------------------------------------
Patrick J. Loll, Ph. D.
Professor of Biochemistry & Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA 19102-1192 USA
(215) 762-7706
pat.l...@drexelmed.edu
