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Look up a standard endotoxin assay (usually an immunoassay) because that's exactly what endotoxin is :-) Artem --- When the Weasel comes to give New Year's greetings to the Chickens no good intentions are in his mind. _____ From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of JOE CRYSTAL Sent: Thursday, February 26, 2009 5:53 PM To: [log in to unmask] Subject: [ccp4bb] Questions regarding Peptidoglycan-binding protein Dear all, Thank you in advance for your expert opinions. I am working on a peptidoglycan (PG)-binding protein, which is composed of two functionally identical domains. Interestingly, when subjected to gel filtration chromatography (Superdex 200), both full-length protein and the single domains migrate as a major peak tailed with a broad shoulder. SDS-PAGE indicates the expected MW for both samples from the major peak and the shoulder, however, with the presence of smears at high molecular weight. Also, after all fractions from both the major peak and the shoulder were combined and incubated at 4 C over 48 hours, the sample was applied to gel filtration again; this time the shoulder actually disappeared (or shifted), resulting in a nice symmetric peak (with the same elution volume as the major peak). My guess is that the protein expressed in E. coli may already specifically or unspecifically associate with PG fragments from cell wall. My question is: how to detect PG fragments in my protein sample? I have tried denaturation-wash-renaturation steps, but I want to know if I have completely got rid of the possible "contaminants". Thank you for suggestions. Joe