Ago2 antibody

Manufactured by Cell Signaling Technology

Sourced in United States, China

The Ago2 antibody is a laboratory reagent produced by Cell Signaling Technology. It is used to detect the Argonaute 2 (Ago2) protein, a key component of the RNA-induced silencing complex (RISC) involved in gene silencing. The Ago2 antibody can be utilized in various analytical techniques, such as western blotting, immunoprecipitation, and immunohistochemistry, to study the expression and localization of Ago2 in biological samples.

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57 protocols using ago2 antibody

AGO2-RIP Assay for circRNA and miRNA Identification

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An Argonaute2-ribonucleoprotein immunoprecipitation (AGO2-RIP) assay was performed on the AGS cells using an RIP kit (RIP-12RXN) (Sigma, St. Louis, MO, USA). The lysed AGS cells were incubated with magnetic beads coated with AGO2 antibodies (Cell Signaling Technology, Boston, USA) or immunoglobulin G, per the manufacturer's instruction. The abundance of hsa_circ_0072309, hsa-miR-34a-3p, and hsa-miR-330-5p in the precipitates were determined by RT-qPCR and gel electrophoresis. The primers used are shown in Table 1. Each assay was performed in triplicate.

Xu B.B., Huang Y., Zheng E.D., Wang J.Y., Zhang C.J., Geng X.G., Wang Y.N, & Pan W.S. (2023). Hsa_circ_0072309 is a prognostic biomarker and is correlated with immune infiltration in gastric cancer. Heliyon, 9(2), e13191.

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Antibody-based Protein Interaction Assay

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PTP1B and α-MHC antibodies were purchased from Abcam. AGO2 antibodies were purchased from Cell Signaling Technology. Anti-Flag antibody, Dynabeads protein A and protein G were from Thermo Scientific. The β-actin and anti-phosphotyrosine, β-MHC PT-66 beads were from Sigma. HRP-conjugated secondary antibodies were from Jackson Laboratories. Protein A/G Plus agarose beads, GAPDH, THRAP1/MED13 and TRβ1 antibodies were from Santa Cruz Biotechnology. HA-HRP antibodies were from Roche. Anti-AGO2 phospho-Tyr³⁹³ antibodies were generated against N²⁸⁹-T-D-P-Yp-V-R-E-F-G-I-M⁴⁰⁰ and affinity-purified at the MRC Protein Phosphorylation and Ubiquitylation Unit (Dundee). Streptavidin-Sepharose beads were purchased from GE Healthcare. Protease inhibitor cocktail tablets and DNase were from Roche. Trizol, cDNA synthesis kit, SYBR Green master mix and TaqMan were from Thermo Scientific. Catalase and superoxide dismutase were from Calbiochem. Surfact-Amps Nonidet P-40, Zeba desalt spin columns and Ni-NTA beads were from Thermo Scientific. RNasin was from Promega. The vectors pET21b-PTP1B(D181A) and pET22b-scFv45 were generous gifts from Dr. Nicholas K. Tonks.

Coulis G., Londhe A.D., Sagabala S.R., Shi Y., Labbé D.P., Bergeron A., Sahadevan P., Nawaito S.A., Sahmi F., Josse M., Vinette V., Guertin M.C., Karsenty G., Tremblay M.L., Tardif J.C., Allen B.G, & Boivin B. (2022). Protein Tyrosine Phosphatase 1B Regulates miR-208b-Argonaute 2 Association and Thyroid Hormone Responsiveness in Cardiac Hypertrophy. Science signaling, 15(730), eabn6875.

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Protein Expression Analysis Protocol

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Proteins were extracted from a cell line as well as adipose tissues using RIPA lysis buffer and the bicinchoninic acid protein assay kit (Sigma) was used to determine protein concentrations. The extracts were resolved via 12% SDS-PAGE, and transferred to PVDF membranes. After blocking for 1 h, the membranes were incubated with the primary anti-EZH2, C/EBP-α, PPAR-γ or AGO2 antibodies (1:000; Cell Signaling Technology, Danvers, MA, USA) at 4ºC overnight, followed by a 2 h incubation with HRP-conjugated secondary antibody at room temperature. The immunoreactive bands were visualized using ECL and normalized to GAPDH (the internal control).

Liu Y., Liu H., Li Y., Mao R., Yang H., Zhang Y., Zhang Y., Guo P., Zhan D, & Zhang T. (2020). Circular RNA SAMD4A controls adipogenesis in obesity through the miR-138-5p/EZH2 axis. Theranostics, 10(10), 4705-4719.

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Anti-AGO2 RIP Assay Protocol

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As reported by a previous study (31 (link)), we also used the Magna RIP RNA-Binding Protein Immunoprecipitation kit (Millipore, Billerica, USA) to conduct the anti-AGO2 RIP assay. Extracts of the treated SK-MES-1R and NCI-H226R cells in RIP buffer were incubated with normal rabbit IgG (Proteintech Group, Inc.; Wuhan, China) and AGO2 antibodies (Cell Signaling Technology), which were combined with magnetic beads. We isolated the immunoprecipitated RNAs and examined genes using RT-qPCR.

Liu J., Jiang M., Guan J., Wang Y., Yu W., Hu Y., Zhang X, & Yang J. (2022). LncRNA KCNQ1OT1 enhances the radioresistance of lung squamous cell carcinoma by targeting the miR-491-5p/TPX2-RNF2 axis. Journal of Thoracic Disease, 14(10), 4081-4095.

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Immunoprecipitation of Ago2 from Mouse Brain

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CA3-DG subfields from individual mice were obtained by microdissection under a microscope and pools of 4–5 were homogenized in an immunoprecipitation buffer and centrifuged. Five micrograms of Ago2 antibody (Cell Signaling Technology, Danvers, MA, USA), was added to 400 μg of the supernatant, vortex-mixed and incubated overnight at 4 °C. Then, 20 μl of 50% Protein-A/G-agarose bead solution (Santa Cruz Biotechnology, Heidelberg, Germany) was added, mixed and incubated. The beads were centrifuged and the pellet washed followed by Trizol extraction27 (link).

Jimenez-Mateos E.M., Arribas-Blazquez M., Sanz-Rodriguez A., Concannon C., Olivos-Ore L.A., Reschke C.R., Mooney C.M., Mooney C., Lugara E., Morgan J., Langa E., Jimenez-Pacheco A., Silva L.F., Mesuret G., Boison D., Miras-Portugal M.T., Letavic M., Artalejo A.R., Bhattacharya A., Diaz-Hernandez M., Henshall D.C, & Engel T. (2015). microRNA targeting of the P2X7 purinoceptor opposes a contralateral epileptogenic focus in the hippocampus. Scientific Reports, 5, 17486.

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AGO2-Mediated RNA Immunoprecipitation

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RNA immunoprecipitation assays were performed by using the Imprint RNA Immunoprecipitation Kit (Sigma, St. Louis, USA) along with the AGO2 antibody (Cell signaling, Rockford, USA). The AGO2 antibody was then recovered by protein A/G beads. XIST, iASPP and miR-140/miR-124 RNA levels in the immunoprecipitates were measured by qRT-PCR.

Liang S., Gong X., Zhang G., Huang G., Lu Y, & Li Y. (2017). The lncRNA XIST interacts with miR-140/miR-124/iASPP axis to promote pancreatic carcinoma growth. Oncotarget, 8(69), 113701-113718.

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Ago2-Mediated RNA Immunoprecipitation

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RIP assay was utilized with Magna RIP RNA-Binding Protein Immunoprecipitation Kit (catalog number: 17–701; Merck Life Science, Shanghai, China) [25 (link)]. The transfected cells were dissolved in RIP lysis buffer, and cell lysate was incubated with magnetic beads bound with the Ago2 antibody (catalog number: 2897; Cell Signaling Technology). IgG (catalog number: 14,708; Cell Signaling Technology) was used as a control group. Then, immunoprecipitated RNA was detected with RT-qPCR assay.

Yuan L., Xu H., Guo R., Lu T, & Li X. (2021). Long non-coding RNA ZFAS1 alleviates bupivacaine-induced neurotoxicity by regulating the miR-421/zinc finger protein564 (ZNF564) axis. Bioengineered, 12(1), 5231-5240.

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AGO2 Immunoprecipitation and qRT-PCR

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The Imprint RNA Immunoprecipitation Kit (Sigma, St. Louis, USA) was used in RNA immunoprecipitation with the AGO2 antibody (Cell signaling, Rockford, USA). The AGO2 antibody was then recovered by protein A/G beads. HOTTIP, miR-192 and miR-204 RNA levels in the precipitates were measured by qRT-PCR.

Ge Y., Yan X., Jin Y., Yang X., Yu X., Zhou L., Han S., Yuan Q, & Yang M. (2015). fMiRNA-192 and miRNA-204 Directly Suppress lncRNA HOTTIP and Interrupt GLS1-Mediated Glutaminolysis in Hepatocellular Carcinoma. PLoS Genetics, 11(12), e1005726.

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AGO2 RNA Immunoprecipitation Assay

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The RIP assay was implemented with a Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore) in line with the manufacturer’s recommendations. AGO2 antibody was applied for RIP (Cell Signaling Technology, USA) with IgG antibody (Cell Signaling Technology) as the negative control. RT-qPCR analysis was used to determine the abundance of RNAs specifically binding to AGO2.

Liang H., Wang J., Zhang P., Yang W., Yang Y., Zhi Y., Wu W, & Dong X. (2021). Long Non-Coding RNA Duxap8 Facilitates Cell Proliferation and Induces Apoptosis in Colorectal Cancer via miR-519b/ZNF277 Axis. OncoTargets and therapy, 14, 4693-4703.

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Investigating circDLG1-miR-141-3p-CXCL12 Axis

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The RIP assay was conducted using the Magna RIP RNA-bing Protein Immunoprecipitation kit (Millipore, USA) according to the provider’s protocol. In brief, cell lysates were cultured with Dynabeads-coated IgG antibody (Millipore, USA) or AGO2 antibody (Cell Signaling Technology, USA) for 12 h at 4 °C. The purified RNA was subjected to qRT–PCR to detect the enriched circDLG1 and miRNA.
For the luciferase activity assay, potential binding sites were predicted using StarBase v3.0 and TargetScanHuman 7.2. Gastric cells were cotransfected with pGL-luc-circDLG1, pGL-luc-CXCL12 3′-UTR, and miR-141-3p mimics or negative control mimics for 48 h, and luciferase activity was detected using the dual-luciferase reporter assay system (Promega, USA) according to the manufacturer’s instructions.

Chen D.L., Sheng H., Zhang D.S., Jin Y., Zhao B.T., Chen N., Song K, & Xu R.H. (2021). The circular RNA circDLG1 promotes gastric cancer progression and anti-PD-1 resistance through the regulation of CXCL12 by sponging miR-141-3p. Molecular Cancer, 20, 166.

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