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: j-kell...@northwestern.edu
*******************************************
----- Original Message -----
From: R.M. Garavito
To: CCP4BB@JISCMAIL.AC.UK
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: garav...@msu.edu
****************************************************************
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: bert.vandenb...@umassmed.edu
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
>
