Monitoring of IBB coupling to TFPAM-6 and of IBB-TFPAM coupling to plasmid DNA. Interaction of IBB with importin β after covalent coupling with plasmid. The coupling reaction of IBB peptide to TFPAM-6 was monitored by reverse phase chromatography using a C18 column, the compounds were detected by UV absorbance at 220 nm (A). HPLC profiles of starting material (IBB, TFPAM-6) and conjugates obtained after 1 h reaction either at pH 7.5 (R pH 7.5) or at pH 2 (R pH2) at room temperature are shown. The elution peaks of 'R pH 7.5' reaction (R1, R2, R3), 'IBB' (IBB), 'R pH 2' (T1, T2, I, T3) chromatograms were collected and analyzed on 10 % NuPAGE electrophoresis gels followed by silver nitrate staining (B). L : 12 marker (Novex). Binding assays were performed between importin β-GST and IBB / plasmid complexes where IBB peptide was either covalently coupled (covalent) or electrostatically associated (electrostatic) to pXL3031 plasmid (C). The IBB / plasmid ratio was either 10 or 25 (mol / mol). The unbound (1) and bound (2) fractions were analyzed, pXL3031 was used as a control (C). Binding assay was also performed between pXL3031 plasmid or pXL3031-IBB covalently coupled chimera (50 mol IBB / mol plasmid) and importin β-sepharose beads (D). The unbound (chimera: 1c and pXL3031: 1p), bound (chimera: 2c and pXL3031: 2p) fractions, control pXL3031 (Cp) and control chimera (Cc) were analyzed. For these assays (C and D) the analysis were performed on 0.8 % agarose gel stained with ethidium bromide. M: 1 kb DNA ladder (Gibco BRL). C: 500 ng pXL3031 plasmid as a control.