RNA immunoprecipitation (RIP) assays were performed using a modified CLIP protocol (48 (link)). Nuclear fraction was isolated and lysed in 1× PBS, pH 7.4, 150 mM NaCl, 1% Triton-X-100, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail for 30 min on ice. The extracts were treated with DNase I (10 U/μl, Roche, Cat. No. 04716728001) and RNase A (10 mg/ml, Sigma, R-4875) for 1 h at 4°C under rotation. The samples were diluted in binding buffer (1× PBS, pH 7.4, 150 mM NaCl, 1% Triton-X-100) and supplemented with RNase inhibitor. Samples were precleared and 10% of were saved for inputs, while the rest were used for immunoprecipitation using anti-U1A antibody (Abcam, mab55751) or rabbit IgGs (Jackson Immuno Research, 011–000-003). The inputs and immunoprecipitated samples were treated with 50 μg Proteinase K for 30 min at 65°C. RNA was then isolated and used for RT-qPCR analyses that were performed using primers ei5-F and ei5-R for exon5–intron 5 junction, ei4-F and ei4-R for exon4–intron4 junction and ei37-F and ei37-R for exon37–intron37 junction.
Rabbit iggs
Manufactured by Jackson ImmunoResearch
Rabbit IgGs are purified immunoglobulin G (IgG) antibodies derived from rabbit serum. They are a common tool used in various immunological and biochemical applications, such as Western blotting, ELISA, and immunohistochemistry.
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2 protocols using rabbit iggs
1
RNA Immunoprecipitation (RIP) Assay Protocol
2
Co-Immunoprecipitation of XAB2 Protein
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U2OS cells (in 15-cm plates) were treated with DMSO or 1 μM CTP for 1 h and collected by scraping in cold PBS and centrifugation. Cells lysates were prepared by resuspending the cell pellets in IP lysis buffer (20 mM Tris–HCl pH 7.8, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40) supplemented or not with 0.2 mg/ml RNase A and incubation for 30 min at 4°C, with vortexing (10 s) every 10 min. Extracts were collected following centrifugation at 15 000 rpm for 20 min. For co-IPs, extracts (500 μg of proteins) were brought to 300 μl with lysis buffer and 500 μl of IP dilution buffer (20 mM Tris–HCl pH 7.8, 150 mM NaCl, 1 mM EDTA, 0.05% NP-40) were added. Fifty microliter of Dynabeads Protein G (Life Technology) coated with a rabbit anti-XAB2 antibody (abcam, ab129487) or rabbit IgGs (Jackson ImmunoResearch Laboratories) were added and the mixture was incubated for 4 h on a spinning wheel at 4°C. The beads were then washed three times with IP wash buffer (20 mM Tris–HCl pH 7.8, 500 mM NaCl, 1 mM EDTA and 0.05% NP-40) and eluted in 50 mM glycine pH 2.8. The eluted samples were mixed with NuPAGE LSD sample buffer and heated for 10 min at 70°C, followed by separation on a NuPAGE 4–12% Bis–Tris gel (Invitrogen) and immunoblotting.
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