Regarding concentrating detergents w/ MWCO concentrators - may I suggest
the following reference:
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.
Michael Matho wrote:
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 <mailto:pat.l...@drexel.edu>
*To:* CCP4BB@jiscmail.ac.uk <mailto: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 <mailto: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 <mailto:pat.l...@drexelmed.edu>
