35s methionine

An in vitro protein-protein binding assay was performed as previously described [14 (link)]. pGBKT7-VP1 bait vector and pGADT7-MAT1 prey vector were used as templates to transcribe and translate in vitro, then labeled with ³⁵S-Methionine (Amersham Pharmacia Biotech) in vitro transcription-translation system (Promega), respectively, to obtain ³⁵S-labeled fusion proteins HA-MAT1 and c-Myc-VP1. The ³⁵S-Methionine-labeled HA-MAT1 and c-Myc-VP1 were incubated at room temperature, then incubated with antibody against c-Myc (Clontech) in lysis buffer, subsequently mixed with protein A/G plus-agarose (Thermo Fisher, USA) and incubated for 3 h at 4°C. The beads were washed three times with lysis buffer. The radioactive antibody–protein complexes were eluted, and subjected to SDS/PAGE and then autoradiography.

We performed a TNT quick-coupled in vitro transcription-translation system (Promega) to obtain proteins used in protein-protein binding assays according to the manufacturer’s procedures. ³⁵S-Methionine (Amersham Pharmacia Biotech) was included in the reaction for the purpose of labeling the synthesized proteins. The templates were pGBKT7-VP1 and pGADT7-GM130. The protocol of the In Vitro protein-protein binding Assay has been previously described45 (link). ³⁵S-Methionine-labeled HA-GM130 and c-Myc-VP1 were incubated with monoclonal anti-c-Myc antibody (Clontech) in lysis buffer (50 mM Tris–HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, 1 mM phenylmethylsulfonyl fluoride), followed by adsorption to BSA blocked protein A/G plus-agarose (Santa Cruz). The beads were washed thrice with lysis buffer (50 mM Tris–HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 0.1% NP-40). The antibody–protein complexes were eluted and then resolved in 10% SDS–PAGE and subjected to autoradiography.