Lysates were prepared by lysing A549 and H460 cells (0°C, 30 minutes) using RNA immunoprecipitation (RIPA) buffer. For RIP experiments, lysates were incubated with antibody against Argonaute 2 (anti‐Ago2, 1:500, MA5‐23515) or isotype IgG (anti‐IgG, 1:1000, 05–4500, all from Invitrogen) and Protein A/G Magnetic Beads (Thermo Fisher Scientific) overnight at 4°C. For RNA pull‐down experiments, lysates were incubated with biotinylated circLDB2 probe (WT‐circLDB2 probe), biotinylated circLDB2 mutant probe in the target region (MUT‐circLDB2 probe), or negative control oligo probe (all from Ribobio) and streptavidin beads (Invitrogen) overnight at 4°C. In both experiments, bound RNA was harvested from beads and used for the assessment of circLDB2 and miR‐346 levels.
Anti ago2
Manufactured by Thermo Fisher Scientific
Anti‐Ago2 is a laboratory reagent used to detect the presence and distribution of the Argonaute 2 (Ago2) protein, a key component of the RNA-induced silencing complex (RISC). It functions as a reliable tool for studying post-transcriptional gene regulation and RNA interference (RNAi) mechanisms.
Automatically generated - may contain errors
Lab products found in correlation
Protein a g magnetic bead, by Thermo Fisher Scientific (1 mentions)
Streptavidin beads, by Thermo Fisher Scientific (1 mentions)
Western lightning plus ecl, by PerkinElmer (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Magna rna binding protein immunoprecipitation kit, by Merck Group (1 mentions)
Proteinase k, by Solarbio (1 mentions)
Protein a magnetic beads, by Thermo Fisher Scientific (1 mentions)
Dna purification kit, by Qiagen (1 mentions)
Anti h3k27ac antibody, by Abcam (1 mentions)
4 protocols using anti ago2
1
Profiling circLDB2 and miR-346 in Cancer Cells
2
Western Blot Analysis of Protein Expression
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IPs were done as described above (excluding SARS-CoV-2 infection), and supernatants were mixed with 4× sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer. Typically, 25 µL (corresponding to ∼40 µg total protein) were separated on a 10% SDS-PAGE gel and electrotransferred to a poly(vinylidene difluoride) membrane (Bio-Rad). After blocking with 5% milk in 1× TBST (20 mM Tris [pH 7.5], 150 mM NaCl, and 0.1% Tween 20), the membrane was probed with the appropriate antibodies and detected with Western Lightning Plus-ECL (PerkinElmer) using a Gbox (Syngene). Primary antibodies used were anti-FLAG M2 (Sigma Millipore), anti-Ago2 (MA5-23515; Invitrogen), and anti-GAPDH (Cell Signaling).
3
RNA-Binding Protein Immunoprecipitation for TUG1 Expression
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An RIP assay was performed to determine whether TUG1 was expressed in the RNA-induced silencing complex using a Magna RNA-binding protein immunoprecipitation kit (EMD Millipore). Chondrocytes (2.0x105 cells) were collected, lysed in RIP buffer (Beijing Solarbio Science & Technology Co., Ltd.) and incubated with magnetic beads (cat. no. 88847; Thermo Fisher Scientific, Inc.) conjugated with anti-argonaute-2 (anti-Ago2; cat. no. ab32381; Abcam; 2 µg/ml) or anti-immunoglobulin G (IgG; cat. no. ab133470; Abcam; 1:5,000) overnight at 4˚C following incubation with Proteinase K (Beijing Solarbio Science & Technology Co., Ltd.) at 55˚C for 30 min. Purified RNA was isolated from the magnetic beads using RNAiso Plus (Takara Biotechnology Co., Ltd.) and analyzed via the aforementioned RT-qPCR assay to determine TUG1 and miR-17-5p enrichment.
4
Chromatin Immunoprecipitation and Sequencing
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After wash twice with 1 × PBS, cells were fixed with 1% formaldehyde (Sigma; F8775) for 10 min at room temperature and stopped with 0.125 M glycine solution for 10 min. Then, cells were collected and lysed in cell lysis buffer (10 mM HEPES pH 7.9, 0.5% NP‐40, 1.5 mM MgCl2, 10 mM KCl, and 1 × cocktail) for 15 min on the ice. Nucleus was extracted and dissolved in nuclear lysis buffer (50 mM Tris–HCl pH 8.1, 10 mM EDTA, 0.3% SDS, and 1× cocktail). After sonication, chromatin fragments were incubated with anti‐H3K27ac antibody (Abcam; ab177178) or anti‐AGO2 (Thermo Fisher Scientific; PA5‐117725) antibody and Protein A magnetic beads (Invitrogen; 10002D) overnight at 4°C. The immunoprecipitated DNA was purified with DNA Purification Kit (Qiagen; 28106). The concentration of ChIP‐derived DNA was detected by Qubit 2.0 and for next‐generation sequencing. Then, quantitative PCR was carried out to verify the ChIP‐seq data and normalized to input DNA (see Table S4 for primer sequences).
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