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 -----Original Message----- From: CCP4 bulletin board on behalf of Jose Antonio Cuesta Seijo Sent: Tue 8/4/2009 12:24 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] detergent crystals Hi Parveen. DDM crystals can look a bit like that. Like a pencil that has been sharpened from both ends until there is very little pencil left, but often the ends are flatter than that. You can get tons of these (also with DM) in conditions with medium weight PEGs. Of course many proteins will also crystallize as hexagonal rods with PEGs :-) I'll take this chance to shre a thought with the community. When we say "contains x% of detergent y", are we sure of what there is in the protein stock? Most membrane protein stocks for crystallization will undergo a concentration step in a filter, and the membrane proteins themselves can be excellent detergent concentrators. For example, 0.05% DDM is about 1 mM DDM, hardly enough to make a belt around proteins that are 100 uM or higher. Do people measure how much detergent there really is in the final crystallization stock? It doesn't seem to get published, instead the concentration in the last purification step is often mentioned. Jose. "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 ***************************
