pET-21a-IFNT

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Recombinant bovine IFNT was produced by Escherichia coli. Briefly, bovine IFNT cDNA (bTP-509A, gifted by Dr R M Roberts, University of Missouri, Columbia, MO, USA) was inserted into pET-21a (Invitrogen Corp.) E. coli expression vectors (Imakawa et al. 1987).	After purification of crude IFNT by HPLC, we confirmed low levels of endotoxin using the LAL Endotoxin Assay Kit (GenScript, Piscataway, NJ, USA). The activity of IFNT was then determined by a viral resistance assay using Madin-Darby bovine kidney cells and was found to be 59 050 IU/ml (5.95×105 IU/mg at 456 μM).

Protocol tips
Recombinant bovine IFNT was produced by Escherichia coli. Briefly, bovine IFNT cDNA (bTP-509A, gifted by Dr R M Roberts, University of Missouri, Columbia, MO, USA) was inserted into pET-21a (Invitrogen Corp.) E. coli expression vectors (Imakawa et al. 1987).
Downstream tips
After purification of crude IFNT by HPLC, we confirmed low levels of endotoxin using the LAL Endotoxin Assay Kit (GenScript, Piscataway, NJ, USA). The activity of IFNT was then determined by a viral resistance assay using Madin-Darby bovine kidney cells and was found to be 59 050 IU/ml (5.95×105 IU/mg at 456 μM).

Publication protocol

Recombinant bovine IFNT was produced by Escherichia coli. Briefly, bovine IFNT cDNA (bTP-509A, gifted by Dr R M Roberts, University of Missouri, Columbia, MO, USA) was inserted into pET-21a (Invitrogen Corp.) E. coli expression vectors (Imakawa et al. 1987). After purification of crude IFNT by HPLC, we confirmed low levels of endotoxin using the LAL Endotoxin Assay Kit (GenScript, Piscataway, NJ, USA). The activity of IFNT was then determined by a viral resistance assay using Madin-Darby bovine kidney cells and was found to be 59 050 IU/ml (5.95×105 IU/mg at 456 μM).

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