@article{mbs:/content/journal/jmm/10.1099/00222615-30-1-69, author = "Kamiya, S. and Reed, P. J. and Borriello, S. P.", title = "Purification and Characterisation of Clostridium Difficile Toxin A by Bovine Thyroglobulin Affinity Chromatography and Dissociation in Denaturing Conditions with or Without Reduction", journal= "Journal of Medical Microbiology", year = "1989", volume = "30", number = "1", pages = "69-77", doi = "https://doi.org/10.1099/00222615-30-1-69", url = "https://www.microbiologyresearch.org/content/journal/jmm/10.1099/00222615-30-1-69", publisher = "Microbiology Society", issn = "1473-5644", type = "Journal Article", abstract = "Summary Highly purified toxin A of Clostridium difficile was obtained by bovine thyroglobulin affinity chromatography followed by two sequential anion-exchange chromatography steps on Q Sepharose FF and Mono Q. After Q Sepharose FF chromatography of a thyroglobulin affinity-purified toxin A preparation, two major peaks of cytotoxicity representing toxins A and B were detected. The homogeneity of the final toxin A preparation obtained from Mono Q anion-exchange fast protein liquid chromatography was ascertained by gel electrophoresis developed by silver stain. The mol. wt of toxin A in non-denaturing conditions was estimated to be 520-540 Kda by native polyacrylamide gel electrophoresis (PAGE) developed by silver stain. In contrast, with sodium dodecyl sulphate (SDS)-PAGE under reducing or non-reducing conditions, a major band of 240 Kda and 10 minor and 27 faint bands (non-reduced conditions), or four minor and 31 faint bands (reduced conditions) were detected after silver staining. In two-dimensional PAGE, the seven minor bands of >240 Kda obtained by non-reducing SDS-PAGE migrated to the 240-Kda position after reduction with β-mercaptoethanol.", }