Dcp1, GW182, Ago2 or RBM4 protein complexes in whole cell lysates or cytoplasmic/nuclear extracts were immunoprecipitated as described previously [18 (link)]. Briefly, cytoplasmic or nuclear extracts were pre-cleared by incubation with pre-blocked protein G-agarose beads for 1 h at 4°C. Beads were pre-blocked by incubation for 1 hr with 100 µg/ml of bovine serum albumin. Pre-blocked beads were washed with buffer C (250 mM sucrose, 10 mM Tris-HCl [pH 7.5], 25 mM KCl, 5 mM MgCl2, 2 mM DTT, 30 U/ml RNase inhibitor, and 1x protease inhibitor cocktail). Protein extract was then centrifuged at 2,000 rpm for 5 min and supernatant (900 µl) was added to 100 µl of pre-blocked protein G-agarose beads that were coated with 10 µl antibody against human Ago2 (clone #4G8; Wako, Richmond, VA), Dcp1 (Genway, San Diego, CA), RBM4 (Abcam, Cambridge, MA), GW182 or IgG isotype control (Santa Cruz Biotechnology, Santa Cruz, CA). After overnight rotation at 4°C, the beads were centrifuged and washed three times with buffer C. Aliquots of bound protein complexes were used for protein analysis by western blotting, and the remainder was used to isolate the bound mRNA and miRNA. The RNA was then analyzed by PCR.
Ago2 clone 4g8
Manufactured by Fujifilm
Sourced in Japan
The Ago2 (clone #4G8) is a research tool used in the scientific community. It functions as a monoclonal antibody that specifically binds to the Ago2 protein, a key component of the RNA-induced silencing complex (RISC). This product is intended for research use only and its precise applications should be determined by the end-user.
Automatically generated - may contain errors
2 protocols using ago2 clone 4g8
1
Immunoprecipitation of Protein Complexes
2
Immunoprecipitation of AGO Proteins
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The control and miR-S1-expressing BxPC-3 cells (7 × 105 cells/2 mL/well) were seeded onto six-well microplates and preincubated overnight at 37 °C. The next day, cells were treated with 100 µM 4-thiouridine (Abcam, Cambridge, UK) for 16 h and exposed to 365 nm UV (0.15 J/cm2). After that, cross-linked cells were lysed with IP lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM KCl, 0.5% NP-40, 20% glycerol, 1 mM NaF, 0.5 mM DTT, and 2 mM EDTA) supplemented with cOmplete ULTRA Tablets (Roche, Basel, Switzerland). After preclearance, these lysates were treated with 10 µg of monoclonal anti-Ago1 (clone 2A7, Wako, Osaka, Japan), Ago2 (clone 4G8, Wako, Osaka, Japan), Ago3 (clone 1C12, Wako, Osaka, Japan), and Ago4 (clone EPR23799-22, Abcam, Cambridge, UK) or isotype mouse IgG or rabbit IgG (Wako) for 2 h at 4 °C. Immunocomplexes were then precipitated with Protein G-conjugated DynabeadsTM magnetic beads (Invitrogen) for 2 h at 4 °C, and RNAs from the resultant precipitate were isolated with ISOGEN (Nippon Gene, Osaka, Japan) and dis-solved with 15 µL of RNase-free H2O.
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