Pull-down analysis
The full-length cDNAs of ApNMV 1a and MdBT2 were amplified and inserted
into pGEX-4T-1 and pET-32a vectors, respectively. The constructs were
then transformed into Escherichia coli BL21 (DE3) to induce the
production of GST- and -HIS tagged fusion proteins under treatment of
isopropyl β-D-1-thiogalactopyranoside at 28 °C for 6 h. The GST-1a and
MdBT2-HIS fusion proteins were premixed for 1 h at 4 °C with gentle
shaking. The protein mix was combined with the glutathione
(GSH)-attached beads (BEAVER), and then incubated at 4 °C for 1 h with
gentle shaking. After washing the beads for five times to remove the
non-specific proteins, the binding proteins were eluted from the
magnetic separated agarose beads with GSH solution and boiled for 5 min.
The supernatant was then used for sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) and immunoblotted with specific
antibodies.
For competitive pull-down assay, same amount (2 μg) of
GST-2apol and 1a-HIS were mixed with simple HIS-tag
protein (2 μg, served as control) or different concentrations (2 μg and
6 μg) of MdBT2-HIS fusion protein, and premixed at 4 °C for 1 h with
gentle shaking. Then following the protocol of pull-down assay described
previously, the binding proteins were resuspended in GSH solution and
used for SDS-PAGE, then detected by immunoblotting with anti-GST
(Abmart) and anti-HIS (Abmart) antibodies.