Glutathione sepharose 4b bead column

GST pull-down assays were conducted as previously described with slight modifications [39 (link)]. Briefly, the encoded GST- or His-tagged fusion proteins and the control GST proteins were expressed in BL21 cells after induction with 0.1 mmol/L IPTG overnight at 18°C. Centrifuged cells were resuspended in lysis buffer (1 × PBS, 0.2 mM PMSF, 1% Triton X-100) and sonicated for 15 min. After centrifugation, the supernatant was applied to a Glutathione–Sepharose 4B bead column (GE Healthcare) or ProteinIso Ni-NTA Resin (TransGen Biotech, China), in accordance with the manufacturers’ instructions. Purified GST/His-tagged fusion proteins were diluted with 1× PBS and filtered through Amicon Ultra 0.5 ml filters (Millipore). Then, 1 μg of purified GST protein or GST fusion protein was captured by the Glutathione–Sepharose 4B beads (GE Healthcare), and His-tagged fusion protein was added for incubation overnight at 4°C. The beads were then washed three times with ice-cold PBS. The supernatant was loaded onto gels, followed by immunoblotting analysis.

GST pull-down assays were conducted as previously described with slight modifications [55 (link)], Briefly, the encoded GST- or His-tagged fusion proteins and the control GST proteins were expressed in BL21 cells after induction with 0.1 mmol/L IPTG overnight at 18°C. Centrifuged cells were resuspended in lysis buffer (1 × PBS, 0.2 mM PMSF, 1% Triton X-100) and sonicated for 15 min. After centrifugation, the supernatant was applied to a Glutathione–Sepharose 4B bead column (GE Healthcare Bio-Sciences) or ProteinIso Ni-NTA Resin (TransGen Biotech, China), following the manufacturer’s instructions. Purified GST/His-tagged fusion proteins were diluted with 1× PBS and filtered through Amicon Ultra 0.5 ml filters (Millipore). Then, 1 μg of purified GST protein or GST fusion protein (GST–PB1) was captured by Glutathione–Sepharose 4B beads (GE Healthcare) for the GST pull-down assays, and His-tagged fusion protein was added overnight at 4°C. The beads were then washed three times with ice-cold PBS. The supernatant was loaded onto gels, followed by immunoblotting with anti-His antibody.