2.2 × 106 F11 cells were plated per 10 cm plate and treatments were conducted the following day after cells had achieved 70–80% confluency (For primary DRG neurons, cells from 6 animals were plated per poly-D-lysine coated 10 cm plate and cultured for 6 days). Cells were treated with vehicle (DMSO) or nelfinavir for 1 h, followed by 100 µM emetine for 5 min. Cells were washed with ice-cold PBS (supplemented with 100 µM emetine), lysed with polysome lysis buffer A (25 mM Hepes-KOH, pH 7.2, 110 mM KOAc, 2.5 mM Mg(OAc)2, 1 mM EGTA, 1 mM DTT, DNase I, 40 U/ml RNasin Plus RNase Inhibitor (Promega), 0.015% digitonin, supplemented with Pierce Protease Inhibitor and Pierce Phosphatase Inhibitor (Thermo) and 100 µM emetine), and removed from the plate with a cell scraper. Crude lysates were collected and centrifuged at 13,000 × g for 10 min at 4 °C to remove debris. The clarified lysate was then loaded onto 0.5 ml 30% sucrose cushion (20 mM Tris pH 7.5, 2 mM Mg(OAc)2, 150 mM KCl, 30% w/v sucrose, supplemented with RNaseIN Plus RNase inhibitor (Promega)). Ribosomes were pelleted by ultracentrifugation at 120,000 × g for 24 h at 4 °C using a Beckman Coulter S55A fixed-angle rotor. Pellets were resuspended in polysome lysis buffer.
Rnasein plus rnase inhibitor
Manufactured by Promega
RNaseIN Plus RNase inhibitor is a recombinant RNase inhibitor protein designed to protect RNA from degradation by RNase enzymes during RNA isolation, purification, and downstream applications. It functions by binding to and inhibiting the activity of RNase enzymes.
Automatically generated - may contain errors
4 protocols using rnasein plus rnase inhibitor
1
Isolation and Purification of Ribosomes
2
Affinity Purification of Stalled Ribosomes
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All in vitro translation analysis were performed using Rabbit Reticulocyte Lysate System, Nuclease Treated (Promega). Translation with generated mRNA was at 30 °C for 15 min unless indicated otherwise in the Fig. legend. The stalled RNCs on the Xbp1u mRNA were affinity purified using the His6-tag on the nascent polypeptide chain and magnetic beads. After the translation reaction, RRL was adjusted to 750 mM KOAc, 25 mM Mg(OAc)2, 250 mM Sucrose, and 2 mM Spermidine, and transferred on ice to stop the reaction. The lysate was applied to Dynabeads for His6-tag isolation and pulldown (Invitrogen) and buffer 7 (50 mM HEPES pH 7.4, 750 mM KOAc, 25 mM Mg(OAc)2, 250 mM Sucrose, 5 mM 2-mercaptoethanol, 0.01% NP-40, 2 mM Spermidine, RNasein Plus RNase Inhibitor (Promega)) at 4 °C for 10 min. After washing by buffer 7 for five times, tethered RNCs were eluted with buffer 8 (50 mM HEPES pH 7.4, 100 mM KOAc, 25 mM Mg(OAc)2, 250 mM Sucrose, 5 mM 2-mercaptoethanol, 0.01% NP-40, 2 mM Spermidine, RNasein Plus RNase Inhibitor) containing 300 mM Imidazole at 20 °C for 15 min.
3
Fluorescent In Situ Hybridization Protocol
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HEK293T cells or HeLa cells grown on coverslips were fixed with 4% paraformaldehyde containing 0.5% Triton X-100 in PBS for 15 min. After rinsing with PBS, the cells were then permeabilized with 0.5% Triton X-100 in PBS for 20 min. Samples were subsequently immersed in 20% glycerol in PBS for more than 30 min and subjected to freeze-thawing four times using liquid nitrogen. Subsequently, the cells were incubated in 0.1 N HCl for 15 min, in 0.1 mg/ml RNase A in 2×SSC for 30 min and in 50% formamide in 2×SSC for 30 min. For denaturation and hybridization, the cells were incubated in a hybridization mixture (2×SSC, 50% formamide, 10% dextran sulfate, 1 mg/ml tRNA and 5 μg/ml probe DNA) at 85 °C for 5 min, and then incubated overnight at 37 °C. After hybridization, the cells were washed with 2×SSC and 50% formamide at 37 °C for 5 min and 2×SSC again for 5 min. RNA FISH was performed using the same protocol as DNA FISH except for the DNA denaturation step. For RNA FISH, 10 units/ml of RNasein Plus RNase inhibitor (N2611, Promega) was added to each incubation step.
4
Polysome Isolation from Cultured Cells
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2.2 x 10 6 F11 cells were plated per 10 cm plate and treatments were conducted the following day after cells had achieved 70-80% confluency (For primary DRG neurons, cells from 6 animals were plated per poly-D-lysine coated 10 cm plate and cultured for 6 days). Cells were treated with vehicle (DMSO) or nelfinavir for 1 hour, followed by 100 µM emetine for 5 minutes. Cells were washed with ice-cold PBS (supplemented with 100 µM emetine), lysed with polysome lysis buffer A (25 mM Hepes-KOH, pH 7.2, 110 mM KOAc, 2.5 mM Mg(OAc)2, 1 mM EGTA,1 mM DTT, DNase I, 40U/ml RNasin Plus RNase Inhibitor (Promega), 0.015% digitonin, supplemented with Pierce Protease Inhibitor and Pierce Phosphatase Inhibitor (Thermo) and 100 µM emetine), and removed from the plate with a cell scraper. Crude lysates were collected and centrifuged at 12,000 rpm for 10 minutes at 4°C to remove debris. The clarified lysate was then loaded onto 0.5 ml 30% sucrose cushion (20 mM Tris pH 7.5, 2mM Mg(OAc)2, 150 mM KCl, 30% w/v sucrose, supplemented with RNaseIN Plus RNase inhibitor (Promega)). Ribosomes were pelleted by ultracentrifugation at 120,000 x g for 24 hours at 4°C using a Beckman Coulter S55A fixed-angle rotor. Pellets were resuspended in polysome lysis buffer.
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