Agree. two comments for your reference: 1. When you have glycerol in your protein buffer, always add same % glycerol in your reservoir solution.You have 10% glycerol in your protein buffer, but not in your reservoir solution, the glycerol will overcome all the other facts and "grasp" water from reservoir to your drop, make your protein more diluted and hard to growing big crystal, try to add 10% glycerol in your reservoir solution to balance the glycerol force, and the vapor will go from drop to reservoir as normal, you may have an other lucky direction.
2. Ni--phosphate crystal will be colored and very hard to form crystal in normal condition, if you worry about Ni treat your sample with EDTA than dialysis against out your protein buffer. Deqian --- On Tue, 11/16/10, Clement Angkawidjaja <[email protected]> wrote: From: Clement Angkawidjaja <[email protected]> Subject: Re: [ccp4bb] how to optimize small rod-shaped crystals To: [email protected] Date: Tuesday, November 16, 2010, 8:19 PM I strongly agree with Eric Larson’s suggestion on trying to see the diffraction of your crystal. The most straightforward solution. Other suggestions may work too, but there are chances they will still give you false positives. If you need bigger crystals, try to slow down the nucleation (use lower temperature, different ratio of protein:crystallant, etc). Clement From: yybbll Sent: Wednesday, November 17, 2010 2:42 AM To: [email protected] Subject: [ccp4bb] how to optimize small rod-shaped crystals 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
