Hi, thank you for you reply. Could you tell me if you try to dye membrane protein crystal with detergent?
Thanks. Y.B. 2010-11-18 yybbll 发件人： Matthew Bratkowski 发送时间： 2010-11-17 02:58:40 收件人： CCP4BB 抄送： 主题： Re: [ccp4bb] how to optimize small rod-shaped crystals I like using Izit dye from Hampton (http://hamptonresearch.com/product_detail.aspx?cid=4&sid=41&pid=33) to check if crystals are protein or salt. If the crystals are protein, the dye should absorb rather readily into the crystals and turn them blue, while the rest of the drop will eventually turn clear. Quite likely, excess dye will also crystallize out as well. Salt crystals will not soak in the dye, and the rest of the drop may remain blue for several days. Using Izit is easy and saves a lot of time. In my experience, I have gotten a lot of false positives from phosphate crystallization conditions, so you want to be sure that the crystals are not salt before you waste any time on optimizing them. Matt On Tue, Nov 16, 2010 at 3:23 PM, Ulli Hain <[email protected]> wrote: If you have a polarizer on your microscope you could see if they are extremely dichroic, in which case they may be salt. You could also open the well up and see if they are heavy - do they sink immediately when you lift them to the top of the drop? This could also indicate salt. But I agree with other posts that they do not seem to small to mount, especially with the new mesh loops they make. -Ulli Adelaide-Ulricke P. Hain Johns Hopkins University Bloomberg School of Public Health Biochemistry & Molecular Biology Quoting yybbll <[email protected]>: > Hi, everybody, > > I try to crystallize one membrane protein. All crystals were grown by > handing-drop vapor diffusion at 20 degree. A protein solution > containing about 8-10mg/ml protein in 20mM Tris (pH7.5), 0.017% DDM, > 100mM NaCl, 10% glycerol, 2mM DDT was mixed with an equal volume of a > reservoir solution containing 45% PEG200, 0.1 M phosphate/citrate > (pH4.2). First crystal appeared in the drop within 4 days. And one > week a lot of crystals appeared in the drops. > > Our question is all of these crystals are too small to check them by > X-ray diffraction and SDS-PAGE. We are not sure they are protein > crystals or salt crystals. Our condition seems difficult to produce > salt crystal. But I am a little warry because we use reloaded our > sample to small Ni-resin column to reduce the concentration of > detergent. Maybe some nickel ion dropped off, and then our protein > sample contained some this ion. And nickel ion may react with > phosphate, and then produced nickel phosphate crystal. Could somebody > tell me if it is possible? > > I attach some photos of our crystals. Could somebody give me some > suggestions about how to optimize this type crystal to get bigger > crystal? > > Thanks a lot! > > Yibin > > > 2010-11-16 > > > > yybbll > > > > ???? Laurie Betts > ????? 2010-11-16 17:13:32 > ???? CCP4BB > ??? > ??? [ccp4bb] expression of Cys-rich small protein > > All - > > We are trying to express for structural studies a 257 AA eukaryotic > intracellular (also possibly nuclear) protein (predicted to be single > domain all-helical) that has 12 Cysteines. No known metal-binding > function not that it couldn't happen. So far (E. coli) it expressed > solubly as MBP fusion (with an N-terminal region deleted predicted > disordered) until cleavage of MBP, then it's not soluble, including > detergents added. THe MBP fusion is usually soluble aggregate so we > assume that our part is not folded right. We have so far assumed it > needs a lot of reducing agent (5 mM DTT or TCEP). Thinking of > trying chaperones and insect cells next. > > Any experience out there that might help? Mostly I wonder about all > the cysteines. Don't really know if that is the problem. > > Laurie Betts >
