α his antibody

The silicon chips were cleaned in oxygen plasma for 2 min at 0.3 mbar and 80% power with the Diener Electronics plasma cleaner (Ebhausen, Germany) followed by gluing the activated surface onto eight-well adhesive slides (ibidi, Planegg/Martinsried, Germany). Afterwards, the chip was washed with ethanol followed by 1× TAE buffer supplemented with 14 mM MgCl₂ pH 8.3. Depending on the experiment, solutions of ^His6EGFP, Rhodamine B dextran (70 kDa), or α-His antibodies were used followed by adding the liposome suspension (1 mg mL^–1, incubation 1 h). Afterwards, the buffer reservoir was washed several times with 1× TAE/14 mM MgCl₂ before the single-transport recordings were carried out. Sealing efficiencies were determined by dividing the EGFP/dye filled cavities by the total cavities per field of view. ^His6EGFP was purified as described^{77 (link)} Rhodamine B dextran (70 kDa) was purchased from Thermo Fisher Scientific (Darmstadt, Germany), and α-His antibody was obtained from Abcam (Berlin, Germany).

Proteins were separated by SDS-PAGE, and transferred to polyvinylidene difluoride membranes (0.45 mm; Amersham Biosciences). The membranes were incubated with primary and secondary antibodies. Amersham ECL Prime Western Blot Detection Reagent (GE Healthcare) was used to visualize the signal. The 22-kD α-zein antibody was used at 1:100, 16-kD-, 27-kD-, 50-kD γ-zein, 14-kD β-zein, 10-kD δ-zein antibodies were used at 1:500. The ZmMADS47, O2 and 19-kD α-zein antibody was used at 1:1000. The α-tubulin (Sigma-Aldrich), α-GST, α-His antibody and secondary antibodies (Abcam) was used at 1:5000.