Glutathione gsh sepharose 4b

Transformed BL21(DE3) E. coli cells were initially cultured in Luria-Bertani medium with 100 µg/mL ampicillin at 37°C 240 RPM until the culture reached an A₆₀₀ of 0.3. GST-LANCL2 was expressed by adding 0.1 mM isopropyl-β-d-thiogalactopyranoside. Induced cells were incubated for 16 h at 20°C 170 RPM. Cells were harvested by centrifugation 45 min 1,559 RCF and lysed by sonication in 50 mM Tris–HCl, pH 8.0, 150 mM NaCl with 0.3 mM Tris(2-carboxyethyl)phosphine (TCEP). Post membrane disruption, lysates were centrifuged at 17,211 RPM for 20 min at 4°C. GST-LANCL2 fusion protein was purified by affinity chromatography using Glutathione (GSH)-Sepharose-4B (GE Healthcare). GST-LANCL2 was eluted from GSH-Sepharose-4B by incubating the resin with 10 mM GSH in 50 mM Tris–HCl, pH 8.0, 150 mM NaCl with 0.3 mM TCEP. GST-LANCL2 proteins were run through a gel filtration column. The fusion proteins were further purified by the AKTA Fast protein liquid chromatography purification systems (GE Healthcare). Protein concentrations were determined by bicinchoninic acid assay. Protein purity was assessed by SDS-PAGE; gels were stained with ProSieve Blue Protein Staining solution.

For immunoprecipitation, Raw264.7 cells or BMDMs were lysed with RIPA buffer containing protease inhibitor cocktail (Roche). Lysates were incubated with a primary antibody overnight at 4°C, followed by incubation with protein A/G agarose (Santa Cruz Biotechnology) for 3hrs at 4°C. Then immunoprecipitates were washed with 1% nonidet P-40 for 3 times. For GST-pull down assay, HEK293T cells were transiently transfected with the indicated plasmids, and at 36hr post-transfection, were lysed with RIPA buffer containing protease inhibitor cocktail (Roche). Lysates were pre-cleared by sepharose 6B (GE Healthcare) for 3hrs at 4°C, followed by incubation with glutathione (GSH) sepharose 4B (GE Healthcare) overnight at 4°C. Precipitated beads were washed with 1% nonidet P-40 for 3 times.

Cytoplasm and membrane protein of HEK293T cells were isolated using the MinuteTM Plasma Membrane Protein Isolation Kit following manufacturer's protocol (Invent sm‐005). For immunoblot analysis, cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris–HCl, 150 mM NaCl, 0.5% sodium deoxycholate, 1% IGEPAL) containing protease inhibitor cocktail (Roche) and phosphatase inhibitor (Na₃PO₄ 1 mM). Whole‐cell lysates were subjected to SDS–PAGE, followed by immunoblotting with the specific antibodies. For pull‐down assay or immunoprecipitation (IP), cell lysates were pre‐cleared by incubation with Sepharose 6B (GE Healthcare) or Protein A/G PLUS‐Agarose (Santa Cruz Biotechnology) for 1 h at 4°C. The pre‐cleared cell lysates were incubated with glutathione (GSH) Sepharose 4B (GE Healthcare), Strep‐Tactin Superflow high‐capacity resin (IBA), or a primary antibody overnight at 4°C, followed by incubation with Protein A/G PLUS‐Agarose (only for IP) for 3 h at 4°C. The immunoprecipitates were washed with lysis buffer for three times before immunoblot analysis.