The RNA-induced silencing complex (RISC) immunoprecipitation (IP) protocol was adapted from that of Keene et al (21 ). Briefly, HCT116 cells were transduced as described above and lysed in polysome lysis buffer (21 ). A mixture of Protein A and Protein G PureProteome magnetic beads (EMD Millipore) were pre-coated with anti-pan AGO antibody (clone 2A8, EMD Millipore) and subsequently mixed with pre-cleared lysate. Following washes, beads were resuspended in NT2 buffer (21 ) supplemented with Proteinase K (Roche Applied Science). Total RNA was extracted using Trizol and the Nucleospin miRNA Purification Kit. Reverse transcription reactions were completed using Transcriptor Reverse Transcriptase using a mixture of oligo-d(T) and random hexamer primers, according to manufacturer’s directions. Quantitative PCR for DPYD was carried out using PrimeTime qPCR primer/probe assay Hs.PT.56a.40723155 (Integrated DNA Technologies) in LightCycler 480 Probes Master enzyme mix.
Anti pan ago antibody clone 2a8
Manufactured by Merck Group
The Anti-pan AGO antibody (clone 2A8) is a laboratory reagent used in research applications. It is designed to detect the presence of Argonaute (AGO) proteins, which are essential components of the RNA-induced silencing complex (RISC). This antibody can be used to identify and quantify AGO proteins in various biological samples, supporting studies related to gene regulation and RNA interference mechanisms.
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2 protocols using anti pan ago antibody clone 2a8
1
RISC Immunoprecipitation for DPYD mRNA Detection
2
RNA-Immunoprecipitation Protocol
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RNA-IP was performed as previously described (Qiu et al., 2017) . Briefly, cells were lysed in a RIP lysis buffer [20 mM Tris-HCl (pH 7.4), 200 mM NaCl, 2.5 mM MgCl 2 , 0.5% Triton X-100, 0.5 U/mL RNase inhibitor (Promega) and a protease inhibitor cocktail (Targetmol)]. After centrifugation, half of the postnuclear lysates were incubated with anti-pan AGO antibody (clone 2A8, EMD Millipore) together with protein-A/G magnetic beads (MCE), and the other half of postnuclear lysates were incubated with protein-A/G magnetic beads (MCE) together with goat anti-mouse IgG (ProteinTech) at 4 C for 24 hrs. Then the immunoprecipitated complexes were washed for 5 times with the same lysis buffer except that NaCl concentration was adjusted to 600 mM. RNAs or proteins were extracted from the complexes and subjected to Northern and Western blottings.
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