Figure 4. His–BCNT doublet in an established clone and its purification.

(A) Exogenous BCNT expressed in transient and established clones. Human BCNT in the pCMV6-AN–His vector was transfected into a derivative of the HEK293T cell line, HEK-TR and the extracts of the transiently expressed cells after 40 h (transfected) or the established clone (G11) were analysed by immunoblotting with anti-BCNT-C Ab (antibody) or anti-His-tag Ab. G* indicates half levels of G11, the extract equivalent to 8 × 10⁴ cells. (B and C) Isolation of His–BCNT from the G11 clone. His–BCNT was purified from an extract of the G11 clone in two ways. The supernatant of the extract was incubated directly with anti-His-tag antibody beads and the adsorbed fraction was eluted with His-tag peptide (B). Alternatively, prior to mixing with the affinity beads, the extract was subjected to ammonium sulfate fractionation and His–BCNT from the dialysates was isolated as in B (C). Each fraction was separated by SDS/PAGE and detected by immunoblot analysis or CBB staining.