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Hi Yibin, I cannot tell the actual scale of your crystals but judging by the curve of the drop outline, these crystals look to be plenty big enough to mount in a loop to stick in the beam and test diffraction. If indeed they are too small to mount a single crystal, and you are mainly interested in testing to see if they are salt, you can gather up several of them in your loop and shoot the group - this won't be useful for data collection if it turns out to be protein but will be sufficient to tell you if your crystals are salt. good luck, Eric __________________________ Eric Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 On Tue, 16 Nov 2010, yybbll wrote: | 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 | | _________________________________________________________________________________________________________________________________________________________ | 发件人： Laurie Betts | 发送时间： 2010-11-16 17:13:32 | 收件人： CCP4BB | 抄送： | 主题： [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 | |