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:[log in to unmask]>
> *To:* [log in to unmask] <mailto:[log in to unmask]>
> *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
>> [log in to unmask] <mailto:[log in to unmask]> 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
>
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>
>
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