The easiest way to measure detergent concentration is to make of use
of surface tension properties - simple, quick and effective. The
method is described in the following paper:
http://www.ncbi.nlm.nih.gov:80/pmc/articles/PMC1367029/
Good luck.
Chris.
2009/10/23 Ezra Peisach <[log in to unmask]>:
> 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
>>
>> [log in to unmask] <mailto:[log in to unmask]>
>>
>>
>
--
Dr. Christopher J. Law,
Lecturer in Biochemistry,
Queen's University Belfast
School of Biological Sciences
Room 185,
Medical Biology Centre,
97 Lisburn Road,
Belfast BT9 7BL.
Tel: 028 9097 2071 (Direct line)
Mob: 07913 078410
Web page: http://www.qub.ac.uk/schools/SchoolofBiologicalSciences/Staff/DrCJLaw/
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