Live-cell single RNA imaging reveals bursts of translational frameshifting

Kenneth Lyon; Luis U. Aguilera; Tatsuya Morisaki; Brian Munsky; Timothy J. Stasevich

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/478040 doi: bioRxiv preprint Fab generation and dye-conjugation Fab generation was done using the Pierce mouse IgG1 preparation kit (Thermo Fisher Scientific) per the manufacturer's instructions. Briefly, beads conjugated with ficin were incubated in 25 mM cysteine to digest FLAG (Wako, 012-22384 Anti DYKDDDDK mouse IgG2b monoclonal) or HA (Sigma-Aldrich, H3663 HA-7 IgG1 mouse monoclonal) antibodies to generate Fab. Fab were separated from the digested Fc region using a NAb Protein A column (Thermo Scientific, product # 1860592). Fab were concentrated to ~1 mg/ml and conjugated to either Cy3 or Alexa Fluor 488 (A488). Cy3 Nhydroxysuccinimide ester (Invitrogen) or A488 tetrafluorophenyl ester (Invitrogen) was suspended in DMSO and stored at -20°C. 100 µg of Fab were mixed with 10 µL of 1M NaHCO3, to a final volume of 100 µL. 2.66 µl of Cy3 (or 5 l of A488) was added to this 100 µL mixture and incubated for 2 hours at room temperature with end-over-end rotation.