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You may unleash a deluge of anecdotes and horror stories, but this is quite common. I have experienced this many times, and you just need to step back and ask yourself what is being done differently: 1. Are all materials used in the preparation of the protein the same (suppliers, sources, expression hosts, purification media, etc.)? I have had this happen several times when I have moved. If you have not rescreened with a broader condition matrix, do so. Slight changes in protein purity and quality (and they are not the same) can radically shift the crystallization behavior. 2. Did you or someone else "improve" the purification? Ultrapure pure protein sometimes crystallizes less well than less pure protein, for a number of reasons. One is that an extra step adding in could remove something critical. In number of cases in my lab, we have solved structures we thought were in the apo-form, but saw that ligands were bound. Making the true apo-form led to getting no crystals. 3. Are you trying to reproduce your work or work of others? If it is the latter, talk to the original people and don't rely on an old notebook. Very subtle differences crop up when doing lab work that impact crystallization, from how you make your PEG or buffer stocks (compared to those acquired in kits) to how someone sets up crystals. One case I know of, the group "lost" their crystals after a technician left. After some investigating, the slight pH difference caused by making up the buffer for a hanging drop reservoir differently was the source of the problem. Hope this helps, 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: [log in to unmask] **************************************************************** On Jul 14, 2014, at 7:44 AM, dusky dew <[log in to unmask]> wrote: > Dear all, > I am trying to reproduce some protein crystals. The protein I am getting after cutting the his tag is very pure. I am using the reported protein concentration. The cofactor and EDTA needs to be added externally. The condition has calcium acetate, peg 4k and sodium acetate buffer. Unfortunately I am getting oil separation and light ppt. I have no clue what is wrong. Please help!