On 8/5/09 5:30 AM, "Jose Antonio Cuesta Seijo" <j...@kemi.ku.dk> 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" <j-kell...@md.northwestern.edu> 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: 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 >>> >> >> >> >> >> > > > -- > *************************** > Jose Antonio Cuesta-Seijo > > Biophysical > Chemistry Group > Department of Chemistry > University of Copenhagen > Tlf: > +45-35320261 > Universitetsparken 5 > DK-2100 Copenhagen, > Denmark > *************************** >
