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The TRAP technique was performed using a modified version of a previous study on hypothalami of mice described under ‘Study design of bacTRAP (EGFPL10a) mice47. One day before TRAP, 375 µl of Dynal Protein G magnetic beads (Invitrogen) was washed three times (1 ml) and resuspended in 275 µl of IP wash buffer (20 mM HEPES (pH 7.4), 150 mM KCl, 5 mM MgCl2 and 1% NP-40). Then, 50 µg of two monoclonal anti-GFP antibodies (HtzGFP-19C8 and HtzGFP-19F7) from the Memorial Sloan-Kettering Cancer Center was added to the magnetic beads and incubated with slow end-over-end mixing overnight at 4 °C.

On the day of TRAP, Dynal Protein G magnetic beads were washed three times (1 ml) and resuspended in 200 µl IP buffer (20 mM HEPES (pH 7.4), 150 mM KCl, 5 mM MgCl2, 1% NP-40, 0.5 mM dithiothreitol and 100 mg ml−1 cycloheximide) to remove unbound anti-GFP. Ice-cold polysome extraction buffer (20 mM HEPES (pH 7.4), 150 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol, 100 mg ml−1 cycloheximide, protease inhibitors and 40 U ml−1 recombinant RNasin Ribonuclease inhibitor) was added to the samples containing mouse hypothalami and homogenized with a motor-driven Teflon glass homogenizer. Homogenates were centrifuged at 4 °C for 10 min at, 2000g to pellet cell debris. Supernatant was transferred to a new microcentrifuge tube and NP-40 (Applichem) and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC; Avanti Polar Lipids) were added to the supernatant at a final concentration of 1% and 30 mM, respectively. After incubation on ice for 5 min, the clarified lysate was centrifuged for 10 min at 13,000g to pellet insoluble material. Next, 30 µl of supernatant (input) was snap frozen in liquid nitrogen until RNA extraction as a comparison to the immunoprecipitated sample. Then, 200 µl of anti-GFP-coated Dynal protein G magnetic beads was added to the supernatant, and the mixture was incubated at 4 °C with end-over-end rotation for 1 h. Beads were subsequently collected on a MagnaRack (Invitrogen), washed four times with high-salt polysome wash buffer (20 mM HEPES (pH 7.4), 350 mM KCl, 5 mM MgCl2, 1% NP-40, 0.5 mM dithiothreitol and 100 mg ml−1 cycloheximide). Input and IP beads were resuspended and incubated in RLT buffer (RNAeasy micro kit, Qiagen) for 5 mins at RT. Supernatant was removed from the IP beads and RNA extraction was performed according to Qiagen’s protocol, including in-column DNase digestion. RNA was resuspended in 10 μl nuclease-free water. RNA quantity and quality of input and IP were determined with a Qubit Fluorometer (Invitrogen) and Agilent 2100 Bioanalyzer.

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