This chapter discusses the nickel chelate chromatography of human immune interferon. Human immune interferon is prepared from peripheral blood lymphocytes, which had been stimulated with staphylococcal enterotoxin A (SEA). Interferon is concentrated by chromatography on the columns of silicic acid and eluted with 0.3 M (CH3)4NCI in phosphate-buffered saline (PBS). In some experiments, silicic acid concentrated interferon is further purified by gel filtration on Ultragel AcA54 eluted with 1.0 M NaCl in PBS before Ni2+chelate chromatography is performed. Interferon is assayed on human WISH cells by estimation of the reduction of cytopathic effect; interferon activity is expressed in terms of National Institutes of Health reference standard. Columns of epoxy activated Sepharose 6B, which have not been previously used, are flushed with 0.05 M EDTA in 1.0 M Sodium Chloride (NaCl) to remove any metal ions, which have been inadvertently bound to the resin. The EDTA is then flushed from the column with Buffer A before charging the resin with Ni2+.
ASJC Scopus subject areas
- Molecular Biology