Protein a g sepharose beads

The IP assay was performed as described previously (27 (link)). Briefly, Cell lysates were then prepared in a RIPA lysis buffer and centrifuged at 12,000 rpm for 10 min at 4 °C, the resultant proteins (200 µg) were first incubated with each Ab of interest (2 µg) for IP at 4 °C overnight and then with protein A/G-Sepharose beads (Beyotime) at 4 °C for 1 hour. The collected beads were then washed 3 times with cold PBS, and finally suspended in 20 µL of 2× loading buffer and boiled for IB with the appropriate Abs.

HEK293T cells were transfected with the overexpression-plasmid for 24 h and then scraped and lysed in RIPA lysis buffer. The lysates were placed on ice for 30 min, following centrifugation at 12,000 g for 20 min. The supernatants were collected into new tubes and incubated overnight with primary antibodies at 4°C. The Protein A/G-Sepharose beads (P2055, Beyotime, China) were added to the overnight incubation tube which was rotated for 2 h at room temperature (RT). Next, the beads were washed four times with cold PBS buffer and boiled in 1 × SDS loading buffer at 100°C for 5 min. For the in vitro pull-down, 1.5 μg GST tagged protein, or 1.5 μg STX11-GST tagged protein was incubated with ATGL-MBP-His protein in protein binding buffer (20 mM Tris-HCl, pH 7.5, 200 mM NaCl,) overnight at 4°C, followed by the addition of GST-beads to pull down the protein complexes. The beads were then washed three times with protein binding buffer before adding 1 × loading buffer. The protein samples were then analyzed by western blotting.