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
***************************