Glutathione agarose 4b

The GST-PbMC1a/1b and His-PbRD21 constructs were introduced into the E. coli BL21 (DE3) strain. One mM isopropyl sulfo-beta-galactoside (IPTG) was added at 37 °C to induce the expression of the fusion proteins. Glutathione-agarose 4B (GE Healthcare) beads and Ni-agarose were used according to the manufacturer’s instructions (GE Healthcare) to purify GST-PbMC1a/1b and His-PbRD21. For the protein pull-down assay, the GST fusion proteins were combined in a glutathione Sepharose 4B column. The loaded matrix was incubated with the purified His fusion protein in binding buffer for 4 h at 4 °C. The beads were centrifuged at 2000 × g for 1 min at 4 °C and washed three times with protein pull-down wash buffer, and the bound proteins were eluted and fractionated by 10% SDS-PAGE.

Heterologous expression and subsequent purification of Spc105 and LaeA proteins were conducted in E. coli BL21 (λDE3) using a combination of GST fusion vector pGEX4T-1 (GE Healthcare) and N-terminal 6X histidine-tag fusion vector pET28a. Briefly, the spc105 gene was inserted into pGEX4T-1 to express GST-Spc105, which was subsequently purified on glutathione-agarose 4B (GE Healthcare) following manufacturer’s recommendations. The laeA gene was cloned into pET28a to yield a His₆-LaeA fusion protein, which was purified using Ni-NTA agarose (GE Healthcare).
GST pull-down experiments were performed according to the manufacturer’s recommendations. Briefly, 20 μg purified GST-Spc105 protein, 25 μl glutathione magnetic beads (Pierce), and 20 μg purified His₆-LaeA protein were co-incubated for 3 h at 4°C in PBS buffer. The magnetic beads were subsequently washed 6 times with PBS buffer and boiled for 10 min in SDS-PAGE loading sample buffer. SDS-PAGE and Western blot analysis were subsequently conducted. Immunodetection of His₆-LaeA was performed using a Mouse His-tag monoclonal antibody at a dilution of 1:5000 (Proteintech) followed by Goat anti-Mouse HRP 1:10,000 (Proteintech).

The GST-AGB1 and His-MPK6 constructs were introduced into E. coli strain BL21 (DE3), inducing the expression of the fusion proteins by adding 1 mM of isopropylthio-β-galactoside (IPTG) at 37°C. The GST-AGB1 and His-MPK6 fusion proteins were purified using glutathione-agarose 4B (GE Healthcare) beads and Ni-agarose, respectively, according to the manufacturer’s instructions (GE Healthcare). For protein pull-down assays, the GST fusion proteins were bound to a glutathione sepharose 4B column and the loaded matrix was then incubated with purified His fusion protein in binding buffer [20 mM HEPES, pH 7.4, 1 mM EDTA, 5 mM MgCl₂, 1 mM DTT, 0.1% Triton X-100, 1 mg ml⁻¹ BSA and 1 mM PMSF] for 4 h at 4°C. The beads were then centrifuged at 2000 g for 1 min at 4°C and washed four times with protein pull-down wash buffer (1 × PBS: 137 mM NaCl, 8.1 mM Na₂HPO₄·12H₂O, 2.68 mM KCl, 1.47 mM KH₂PO₄, 1 mM PMSF, pH 7.4). Bound proteins were eluted, and then fractionated by 10% SDS-PAGE, and analyzed by Western blotting using antibodies of HisProbe-HRP (Invitrogen).