Coupled Transcription-Translation of Radiolabeled AnxA2

The TNT^® T7 Quick for PCR DNA (Promega, Madison, United States) is an in vitro coupled transcription/translation system based on rabbit reticulocyte lysate (RRL) and was supplemented with the cDNA coding for full-length rat anxA2 mRNA (including the UTRs and containing a T7 promoter site; described above) and [³⁵S]-Met (EasyTag™ L-[³⁵S]-Met; 10 mCi/mL; PerkinElmer, Waltham, United States). AnxA2 in 20 mM Tris (pH 8) was added to the RRL before the addition of the cDNA and the RRL constituted 63% of the assays. The reaction was performed as previously described (Strand et al., 2021 (link)). Incorporated [³⁵S]-Met into the AnxA2 protein and total radioactivity in the reaction mixture were measured in a Packard liquid scintillation counter.