RIP assay was performed to explore whether RNA induced silencing complex (RISC) contained NEAT1 and miR-34a-5p in 5-8F cells using an Imprint RNA Immunoprecipitation (RIP) kit (Millipore, Billerica, MA, USA) referring to the directions of the manufacturers. In brief, 5-8F cells transfected with miR-34a-5p mimics were lysed in RIP lysis buffer (Takara, Beijing, China) and were incubated with protein A/G magnetic beads and Argonaute-2 antibody (anti-Ago2, Abcam, Cambridge, UK) or IgG antibody (anti-IgG, Abcam). Then, cell lysates in the miR-34a-5p mimics group were centrifuged. The supernatants were marked as output, and immunoprecipitation complexes were named as IP. Western blot analysis was performed to validate successful RIP with anti-Ago2. Lastly, qRT-PCR assay was used to detect NEAT1 enrichment in the RIP RNA fraction.
Anti argonaute 2 antibody ago2
Manufactured by Abcam
Sourced in United States
Anti-Argonaute 2 antibody (AGO2) is a laboratory reagent used to detect and study the Argonaute 2 protein, a key component of the RNA-induced silencing complex (RISC). This antibody can be used in various applications such as Western blotting, immunoprecipitation, and immunocytochemistry to investigate the expression and localization of Argonaute 2 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (2 mentions)
Imprint rna immunoprecipitation rip kit, by Merck Group (1 mentions)
Anti igg, by Abcam (1 mentions)
Rip lysis buffer, by Bio-Rad (1 mentions)
Anti igg antibody, by Merck Group (1 mentions)
Magna rip kit, by Merck Group (1 mentions)
M 280 streptavidin magnetic bead, by Thermo Fisher Scientific (1 mentions)
Rip rna binding protein immunoprecipitation kit, by Merck Group (1 mentions)
5 protocols using anti argonaute 2 antibody ago2
1
RIP Assay for NEAT1 and miR-34a-5p
2
Profiling circRNAs and miRNAs by RIP-qPCR
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SCC-15 and HSC-3 cells were lysed via RIP lysis buffer (Bio-Rad) at 48 h post transfection. Subsequently, the cell lysate was incubated overnight at 4°C with Protein A/G Magnetic beads (Pierce, Rockford, IL, USA) containing argonaute 2 antibody (anti-Ago2; Abcam, Cambridge, UK) or immunoglobulin G antibody (anti-IgG; Abcam). The RNA enrichments of circGDI2, miR-454-3p and FOXF2 from purified RNA immune-precipitate were tested by qRT-PCR.
3
RIP Assay for Argonaute 2 Interaction
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RIP assay was performed using the Magna RIP Kit (EMD Millipore, USA). Huh-7 and Hep3B cells (1 × 107) were treated with the RIP lysis buffer. The cellular extracts were exposed to magnetic beads coated overnight at 4 °C with anti-IgG antibody (EMD Millipore) or anti-Argonaute 2 antibody (AGO2, #ab32381; Abcam, USA). TRIzol (Thermo Fisher Scientific) was used for RNA extraction. The extracted RNA was analyzed using qRT-PCR.
4
Argonaute-2 Mediated miRNA Profiling
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RIP™ RNA‐Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) was used for RIP according to the manufacturer's protocol. Briefly, cells were lysed using complete RIP lysis buffer and incubated with magnetic beads conjugated with anti‐argonaute‐2 antibody (AGO2, (1:1000; Abcam, San Francisco, USA) or control anti‐IgG antibody for 6 hours at 4°C. After protein removal from the beads, RT‐qPCR analysis of the purified RNA was conducted to verify the presence of target RNA.
Biotin‐labelled miR‐205‐5p‐WT, miR‐205‐5p‐MUT and control probe were purchased from Geneseed Biotech (Shanghai, China). Cells were lysed with lysis buffer and incubated with specific probes. Then, the cell lysates were incubated with M‐280 streptavidin magnetic beads (Invitrogen, San Diego, CA, USA) to pull‐down the biotin‐labelled RNA complex according to the manufacturer's protocol. The bound RNAs were purified using Trizol for qRT‐PCR analysis.
Biotin‐labelled miR‐205‐5p‐WT, miR‐205‐5p‐MUT and control probe were purchased from Geneseed Biotech (Shanghai, China). Cells were lysed with lysis buffer and incubated with specific probes. Then, the cell lysates were incubated with M‐280 streptavidin magnetic beads (Invitrogen, San Diego, CA, USA) to pull‐down the biotin‐labelled RNA complex according to the manufacturer's protocol. The bound RNAs were purified using Trizol for qRT‐PCR analysis.
5
RBP Immunoprecipitation for RNF144A-AS1
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The MagnaRIP RNA Binding Protein Immunoprecipitation Kit (Merck Microwells) was used according to the manufacturer's instructions. The cell lysate was incubated with microspheres coated with 5 µg anti-Argonaute-2 antibody (AGO2) (Abcam, MA, USA) and anti-PUF60 (Abcam), and the control IgG was rotated overnight at 4°C. Then total RNA was extracted, and the expression of RNF144A-AS1 was detected by qRT-PCR.
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