Wouldn't the lack of solubility of the alpha form of DDM suggest an easy purification protocol for the beta form?
JPK On Fri, Apr 27, 2012 at 8:40 AM, R. M. Garavito <rmgarav...@gmail.com>wrote: > Hongjun, > > I am in agreement with Bert as DDM is exceedingly difficult to > crystallize, even in organic solvents. This is one of the reasons it is so > expensive. However, you can produce a lot of "quasi"-crystals that do show > low resolution diffraction. As Bert said, you may have protein/detergent > crystals that are just poorly ordered. I would disagree with Bert only > slightly concerning these crystals in that while you might suppose that > most or all of the lattice contacts are mediated by detergent and not by > protein. Instead, you might also be observing protein-protein contacts > being disturbed by less than optimal detergent contacts (either > detergent-detergent or detergent-protein). Try changing the detergent to > decyl-maltoside (DM) to see if you get similar results. It was the change > from DDM to DM that really gave great crystals for the 13-subunit bovine > cytochrome c oxidase. > > Another thing to watch out for is the dreaded contamination factor, either > by protein or detergent. I have seen cases where crystals were from a > contaminating protein (such as those which may bind to Ni-affinity resin > and are not separated by gel filtration) at as low as 1% (by weight) > contamination. More insidious is detergent contamination. DDM is is the > beta anomer, but all batches are contaminated with varying amounts of the > alpha anomer. The alpha anomers of alkyl glycoside detergents tend to > crystallize much more readily than the beta anomers. Despite their best > efforts, manufacturers occasionally produce batches with a high level > of alpha anomer contamination. I have personally tested a batch of > beta-octyl glucoside (from a very reputable company) that did not dissolve; > other batches from a different company were cloudy when making a 10% stock > solution. Alpha-octyl glucoside is not soluble below ~32C and make some > very nice crystals in aqueous solution at room temperature. So try a batch > of DDM from another source. > > Best of luck, > > Michael > > ****************************************************************** > *R. Michael Garavito, Ph.D.* > *Professor of Biochemistry & Molecular Biology* > *603 Wilson Rd., Rm. 513** * > *Michigan State University * > *East Lansing, MI 48824-1319* > *Office:** **(517) 355-9724 Lab: (517) 353-9125*** > *FAX: (517) 353-9334 Email: rmgarav...@gmail.com* > ****************************************************************** > > > > > On Apr 26, 2012, at 5:32 PM, Van Den Berg, Bert wrote: > > Generally speaking it is quite hard to crystallize DDM since it is so > soluble (>20% in water). You most likely have protein crystals (of course > containing a lot of detergent as well) that are just not ordered, > presumably because most or all of the lattice contacts are mediated by > detergent and not by protein. Unfortunately this is the norm for membrane > protein crystallization. > > Good luck, Bert > ------------------------------ > *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of 于洪军 [ > hongju...@moon.ibp.ac.cn] > *Sent:* Friday, April 27, 2012 6:07 AM > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* [ccp4bb] detergent crystal? > > > Hi, > > I am trying to screen crystals of membrane protein in DDM solutions. I got > crystals and its diffraction pattern as I enclosed. Membrane protein > crystallization seems quite different from soluble protein. The condition > contains PEG400. I learn from other topic here that PEG400 can easily > produce DDM detergent crystals. Is it detergent crystal ? Can I tell this > from the diffraction pattern? Advices would be greatly appreciated. Thank > you. > > > Hongjun > > > -- ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu *******************************************
