On 8/5/09 5:30 AM, "Jose Antonio Cuesta Seijo" <[log in to unmask]> wrote:
> Jakob,
>
> That Compacdt disc appearance is very familiar, those were right next to
> the hexagonal ones in my experiments. And all were oily when touched. It is
> very likely that my "detergent" crystals were in reality detergent-protein
> complexes with less than crystalline order. I was doing experiments with
> very high detergent concentrations (up to 6%) and in some cases the
> crystals would be there in about 50% of the conditions. Their numbers also
> correlated better with the detergent concentration than with the protein
> concentration, but all that is still compatible with protein-detergent
> complexes, of course.
> Regarding measuring the final detergent concentration, a fast method is
> described in :
>
> "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 Volume 323, Issue 2, 15 December 2003, Pages
> 234-241
>
> It uses TLC, and the protein crystallization stock can be spotted directly,
> water and all. The standard can also be in water. In my hands, the crucial
> step was to dry this water thoroughly before running the TLC. I adapted
> this to small TLC plates which can be run in a sealed beaker. Running your
> sample in between appropriate standards will give you an estimation of your
> detergent concentration in as little as 2 hours.
>
> Cheers.
>
> Jose.
>
>
>
> "Jacob Keller" <[log in to unmask]> wrote:
>> A recommendation: try looking at the crystals while rotating the
> polarizers.
>> Often you can get detergent or detergent-protein complex "crystals" which
> have
>> sharp edges, but are actually liquid crystals. This will be manifest as a
>> compact-disc (or vinyl LP, depending on your vintage) appearance which
> rotates
>> in sync with the rotation of the polarizers. Several colleagues and I
> have been
>> plagued with these false positives, which are in our experience extremely
> hard
>> to optimize into real crystals.
>>
>> Another possibility: crystallization with a fluorescent or otherwise
> detectable
>> substrate analogue could also be helpful, at least for determining
> whether there
>> is protein in the sharp-edged objects.
>>
>> The best test, of course, is to mount the objects and put them in the
> x-ray
>> beam.
>>
>> Regards,
>>
>> Jacob Keller
>>
>>
>> *******************************************
>> Jacob Pearson Keller
>> Northwestern University
>> Medical Scientist Training Program
>> Dallos Laboratory
>> F. Searle 1-240
>> 2240 Campus Drive
>> Evanston IL 60208
>> lab: 847.491.2438
>> cel: 773.608.9185
>> email: [log in to unmask]
>> *******************************************
>>
>> ----- Original Message -----
>> From: R.M. Garavito
>> To: [log in to unmask]
>> Sent: Tuesday, August 04, 2009 12:37 PM
>> Subject: Re: [ccp4bb] detergent crystals
>>
>>
>> Parveen,
>>
>>
>> Bert and Pascal are correct in that most alkyl glycoside detergent are
>> notoriously difficult to crystallize in aqueous solution when you have
> the
>> beta-anomer (what we normally buy). However, the alpha-anomers can be
> quite
>> easy to crystallize and can contaminate batches of beta-alkyl glycoside
>> detergents. While the quality control procedures are usually good enough
> to
>> ensure that the alpha-anomer contamination of DDM, DM, and OG are low, it
> may
>> not be low enough for all crystallization experiments. Twenty or so
> years ago,
>> I was even shown a batch of "pure" beta-OG from a company I shall not
> name which
>> was insoluble in water.
>>
>>
>> Some people have complained about this, but the impact of alpha-anomer
>> contamination on crystal growth and spurious detergent crystallization is
>> unknown. If this persists and you are sure that those are detergent
> crystals,
>> you might ask to see information about alpha-anomer contamination for
> your batch
>> of detergent. Companies like Anatrace will be quite forthcoming with
>> information, but larger companies (Sigma or Rohm & Haas) may give you the
> run
>> around.
>>
>>
>> Good luck,
>>
>>
>> Michael
>>
>>
>> ****************************************************************
>>
>> R. Michael Garavito, Ph.D.
>>
>> Professor of Biochemistry & Molecular Biology
>>
>> 513 Biochemistry Bldg.
>>
>> Michigan State University
>>
>> East Lansing, MI 48824-1319
>>
>> Office: (517) 355-9724 Lab: (517) 353-9125
>>
>> FAX: (517) 353-9334 Email: [log in to unmask]
>>
>> ****************************************************************
>>
>>
>>
>>
>>
>> On Aug 4, 2009, at 12:51 PM, Van Den Berg, Bert wrote:
>>
>>
>> Hi Jose,
>>
>> how do you know that those crystals were detergent and not protein?
> My
>> impression is that it is really hard to crystallize DDM, and even harder
> for DM
>> (solubilities > 20% in water). The easiest (?) way to check this may be
> to take
>> some crystals, wash them well and run them out on a PAGE gel. If you
> don't see
>> anything and you've taken enough crystals, then you're probably dealing
> with
>> pure detergent crystals. As for your second point, you're right. For most
>> low-cmc detergents the total detergent concentration will be
> substantially
>> higher than reported, since a substantial amount is always bound to your
>> protein. For 1 mM DDM, you would have only ~ 20 uM micelles, assuming an
>> aggregation # of 50 (its higher). I don't think people measure the total
>> detergent concentration in the end; for maltosides one could in principle
> do a
>> Fehling's based assay to get the concentration.
>>
>> Cheers, Bert
>>
>> Bert van den Berg
>> University of Massachusetts Medical School
>> Program in Molecular Medicine
>> Biotech II, 373 Plantation Street, Suite 115
>> Worcester MA 01605
>> Phone: 508 856 1201 (office); 508 856 1211 (lab)
>> e-mail: [log in to unmask]
>> http://www.umassmed.edu/pmm/faculty/vandenberg.cfm
>>
>>
>> "Parveen Goyal" wrote:
>>> Hi All,
>>>
>>> I got some hexagonal crystals in one of my crystal condition. The
> protein
>>> is
>>> a membrane protein and contains 0.05% DDM. Has anybody seen DDM
> crysals
>>> and > if yes, how do they look like?
>>>
>>> thanks in advance
>>>
>>> Parveen Goyal
>>>
>>
>>
>>
>>
>>
>
>
> --
> ***************************
> Jose Antonio Cuesta-Seijo
>
> Biophysical
> Chemistry Group
> Department of Chemistry
> University of Copenhagen
> Tlf:
> +45-35320261
> Universitetsparken 5
> DK-2100 Copenhagen,
> Denmark
> ***************************
>
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