Tnt quick coupled transcription translation system kit

Flag-TRIM31 and HA-MAVS proteins were expressed with a TNT Quick Coupled Transcription/Translation System kit (Promega, L1171) according to the manufacture’s protocol. His-USP18 protein was bought from (Abiotech, Jinan, China), which was purified with nickel beads. In vitro ubiquitination assays were performed as described previously. Briefly, E1, UbcH5a, HA-MAVS, reaction buffer, ubiquitin, ATP, and MgCl₂ were mixed with or without Flag-TRIM31 and His-USP18. The reaction mix was incubated at 30 °C for 1 h followed by addition of 6× SDS loading buffer at 95 °C for 10 min to stop the reaction.

The tested proteins were expressed with a TNT Quick Coupled Transcription/Translation System Kit (L2080, Promega) following the manufacturer's instructions. For the in vitro ubiquitination assay, 5 nM TRAF6 and 1 μM TAK1 protein were mixed with 100 nM His-E1, 1 μM His-E2 (Ubc13/Mms2) and 2.5 μM Bt-Ub in 50 μM ubiquitination reaction buffer from the ubiquitination kit (BML-UW9920-0001, Enzo Life Sciences) according to the manufacturer's instructions. Samples were subsequently immunoprecipitated with an anti-TAK1 antibody (5,206, Cell Signaling Technology) and separated on SDS–PAGE followed by streptavidin conjugated to HRP (ab7403, Abcam).

The gene to be expressed was cloned downstream of a T7 promoter in the pSGI expression vector
[29 (link)]. The plasmid was then added to the reaction mixture containing T7 polymerase, amino-acid mixture (lacking methionine), appropriate buffer and rabbit reticulocyte lysate using the TNT® quick coupled transcription/translation system kit (Promega Corporation, USA) according to the manufacturer’s guidelines. Exogenously added [³⁵S] methionine/cysteine was used (>1,000 Ci/ml) to label the protein as recommended for 90 min at 30°C. Subsequently, the in vitro synthesized proteins were analyzed by SDS-PAGE and fluorography.