1. Epitope-tagged rice RPL18 assembles into functional ribosomes that can be purified by TRAP. (A) Confirmation of RPL18 assembly into ribosomes.
Epitope-tagged rice RPL18 assembles into functional ribosomes that can be purified by TRAP. (A) Confirmation of RPL18 assembly into ribosomes. The p35S:HF-OsRPL18 rice line was used as the source of ribosomal complexes, which were separated by ultracentrifugation on a 20–60% (w/v) sucrose density gradient. The absorbance at 254 nm was recorded to detect the ribosomal subunits of 40S and 60S, monosomes (80S), and polysomes. The gradient was fractionated and proteins in the 10 fractions were analyzed by SDS-PAGE separation and western blotting processed with anti-FLAG (α-FLAG) or anti-RPS6 (α-RPS6) antisera. Molecular mass markers are indicated on the left. (B) Purification of polysomes by TRAP. Equal weights of pulverized tissue from untransformed Nipponbare (control) and homozygous transgenic p35S:HF-OsRPL18 shoots were solubilized in polysome extraction buffer to obtain a clarified supernatant (total). The extract was incubated with anti-FLAG-bound Dynabeads coupled to Protein G to bind HF-RPL18. The supernatant (unbound fraction) was collected to evaluate the efficiency of the immunopurification. The magnetically captured protein–RNA complexes (TRAP fraction) was eluted from the beads using 3X-FLAG peptide. Each fraction was analyzed by western blot with α-FLAG and α-RPS6. The expected molecular mass of HF-RPL18 is 25 kDa. SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TRAP, translating ribosome affinity purification.
Dongyan Zhao et al. G3 2016;7:
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