Thanks for the help, Here is some more information. Protein is expressed in E. Coli, three methionines in the protein. Protein forms a disulphide linked homo-dimer. It is prepared from the soluble fraction of E. coli lysis buffer. Protein is ~12kDa. Protein runs identical to the native on a non reducing SDS gel.
M. Gordon Joyce, Visiting Fellow, Structural Immunology Section, Laboratory of Immunogenetics, NIH/NIAID, 12441 Parklawn Drive, Rockville, MD 20851 Phone: 301 594 0242 Office 301 496 3792 Lab From: Pascal Egea [mailto:pas...@msg.ucsf.edu] Sent: Sunday, March 28, 2010 6:17 PM To: Joyce, Gordon M. (NIH/NIAID) [F] Subject: Re: [ccp4bb] Se-met protein as soluble aggregate Hi Gordon, You may have to give us more details about your protein. I would first ask you in what host (Ecoli, yeast ...) are you expressing your protein and how many methionines do you have in your protein (and what size of protein). Do you prepare it from a soluble cytoplasmic fraction or a periplasmic fraction for example? It is not unusual to observe changes in solubility and behaviour for a given seleno-protein when compared to its unlabeled equivalent. This is why you usually have to recalibrate your crystallization conditions (or even change them drastically for the seleno-labelled protein). Selenium is overall more hydrophobic than sulfur. Does your seleno-protein run fine on a non-reducing SDS gel when compared to the native protein? If oxidation is the problem you can try to add EDTA and methionine in your IEX and gel filtration buffers. I would also see if switching the GF step before the IEX step changes things. Do you need the IEX step? If agregation is only due to increased hydrophobicity then may be increasing ionic strength in your GF buffer will solve your problem. Alternatively use cobalt chelating resin for affinity purification, it is milder and requires less imidazole at the elution step, just in case imidazole is too aggressive on your protein. I have noticed this in the past on some of my samples. Hope this helps Pascal -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 314 Biomedical Sciences Research Building office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu<mailto:pe...@mednet.ucla.edu>