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Ago (Argonautes) Antibody

Catalog Number
Format
Price
C15200167-100
(MAb-167-100)
100 µg /42 µl
$430.00
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Purified monoclonal antibody prepared by immunizing mice with recombinant human Ago2 protein (GST fusion protein containing amino acids 47–859 of Ago2). The antibody also recognizes other Ago family members although with the highest affinity for Ago2.

Lot003
Concentration2.4 μg/μl
Species reactivityHuman, mouse, other (wide range)
TypeMonoclonal
PurityProtein A purified
HostMouse
PrecautionsThis product is for research use only. Not for use in diagnostic or therapeutic procedures.
Applications Suggested dilution Results
IP (RIP) 8 μg per IP Fig 1
HITS-CLIP - Fig 2
WB 1:500 Fig 3
IF 1:500 Fig 4
IHC 1:500
  • Validation Data

    Ago (Argonautes) Antibody validated in IP

    Figure 1. Immunoprecipitation of endogenous Argonaute proteins using the Diagenode Ago monoclonal antibody
    Hela cells (10 cm dish per IP) were subjected to UV crosslinking, lysed in 70 μl of 1xPXL buffer (1xPBS, 0.1% SDS, 0.5% Nadeoxycholate, 0.5% NP-40) by incubating on ice for 10 min, then treated with DNase I. Dynabeads Protein A (40 μl each) were first incubated with 12 μg anti-mouse IgG, used as a bridge antibody, briefly washed, and then incubated with Diagenode Ago monoclonal antibody (Cat. No. C15200167) (4 or 12 μg), or non-immune mouse IgG control (4 or 12 μg). The cell lysate and Dynabeads were mixed and incubated at 4°C for overnight. The beads were washed twice each at 4°C for 5 min using 500 μl of (i) 1 x PXL buffer, (ii) 5 x PXL (5xPBS, 0.1% SDS, 0.5% Na-deoxycholate, 0.5% NP-40), and (iii) 1 x PNK (50 mM Tris pH7.4, 10 mM MgCl2, 0.5% NP-40). Ten μl of input (10% of starting material), 10 μl of supernatants after immunoprecipitation (10%), and all of immunoprecipitants (~90%) were loaded on SDS-PAGE gel and subjected to western blot analysis using the Diagenode Ago monoclonal antibody (Cat. No. C15200167) and the ECL plus reagent.

    coimmunoprecipitation

    Figure 2. Allelic imbalance Ago-RIP assay with the Diagenode Argonautes monoclonal antibody
    The goal of this experiment is to show the preferential coimmunoprecipitation of the miRNA targeted alleles using Diagenode Ago monoclonal antibody (Cat No. C15200167). Briefly, Hela cells were transfected with: pRL-TK-4A + pRL-TK-4G + pcDNA-miR-1 (miR-1 is supposed to target “A” allele) pRL-TK-4A + pRL-TK-4G + pcDNA-miR-1C (miR-1C is supposed to target “G” allele) pRL-TK-4A + pRL-TK-4G + pcDNA-miR-377 (miR-377 is supposed to target neither allele) Cell lysate was immunoprecipitated with Protein G agarose beads incubated with Diagenode Ago monoclonal antibody. RNA was isolated from the complexes from the input and IP samples, reverse transcribed using random hexamers, and subjected to RT-PCR to confirm enrichment of miRNA targeted allele in IP sample compared to input sample. The PCR products were directly sequenced. The electropherograms at the polymorphic site are shown in the second panel. “The predicted miRNA target alleles were efficiently coimmunoprecipitated by the Diagenode Ago monoclonal antibody (Cat. No. C15200167). More details can be found in Takeda H, Charlier C, Farnir F, Georges M., RNA, 2010,1854-63, Epub 2010, Aug 2. Results were kindly provided by Haruko Takeda (Unit of Animal Genomics, GIGA Research Center, Faculty of Veterinary Medicine, University of Liège, 4000-Liège, Belgium).

    Ago (Argonautes) Antibody validated in WB

    Figure 3. Western blot analysis using the Diagenode Ago monoclonal antibody
    Whole cell extracts (40 µg) from HeLa cells were analysed by Western blot using the Diagenode Ago monoclonal antibody (Cat. No. C15200167) diluted 1:500 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right; the marker (in kDa) is shown on the left.

    Ago (Argonautes) Antibody validated in IF

    Figure 4. Immunofluorescence using the Diagenode Ago monoclonal antibody
    HeLa cells were stained with the Diagenode antibody against Ago (cat. No. C15200167) and with DAPI. Cells were fixed with 4% formaldehyde for 10’ and blocked with PBS/TX-100 containing 1% BSA. The cells were immunofluorescently labelled with the Ago antibody (middle) diluted 1:500 in blocking solution followed by an anti-mouse antibody conjugated to Alexa594. The left panel shows staining of the nuclei with DAPI. A merge of both stainings is shown on the right.

  • Target Description

    Members of the Argonaute (Ago) protein family are central to RISC function. Argonaute proteins bind to mature microRNA (miRNAs) and short interfering RNAs (siRNAs) and form the core components of effector complexes that mediate miRNA and siRNA function. Argonautes are needed for miRNA-induced silencing and contain two conserved RNA binding domains: a PAZ domain that can bind the single stranded 3’ end of the mature miRNA and a PIWI domain that structurally resembles ribonuclease-H and functions to interact with the 5’ end of the guide strand. Some argonautes, for example human Ago2, cleave target transcripts directly; argonautes may also recruit additional proteins to achieve translational repression. The human genome encodes eight Argonaute proteins divided by sequence similarities into two families: AGO (with four members present in all mammalian cells and called E1F2C/hAgo in humans), and PIWI (found in the germ line and hematopoietic stem cells).

  •  Testimonials

    Diagenode Ago monoclonal antibody (Cat No. C15200167) immunoprecipitates the predicted miRNA target alleles very efficiently

    Haruko Takeda, University of Liège, Belgium
  •  Applications
    RIP
    RIP Read more
    IHC
    Immunohistochemistry Read more
    IF
    Immunofluorescence: Diagenode offers huge selection of highly sensitive antibodies validated in IF. Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9 HeLa cells transfected with a Cas9 expression vector (... Read more
    WB
    Western blot : The quality of antibodies used in this technique is crucial for correct and specific protein identification. Diagenode offers huge selection of highly sensitive and specific western blot-validated antibodies. Learn more about: Load... Read more
  •  Documents
    Datasheet Ago C15200167 DATASHEET
    Purified monoclonal antibody prepared by immunizing mice with recombinant human Ago2 protein (GST...
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    Epigenetic Antibodies Brochure BROCHURE
    More than in any other immuoprecipitation assays, quality antibodies are critical tools in many e...
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    Antibodies you can trust POSTER
    Epigenetic research tools have evolved over time from endpoint PCR to qPCR to the analyses of lar...
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  •  Safety sheets
    Ago Argonautes monoclonal antibody SDS GB en Download
    Ago Argonautes monoclonal antibody SDS US en Download
    Ago Argonautes monoclonal antibody SDS BE nl Download
    Ago Argonautes monoclonal antibody SDS BE fr Download
    Ago Argonautes monoclonal antibody SDS FR fr Download
    Ago Argonautes monoclonal antibody SDS ES es Download
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    Ago Argonautes monoclonal antibody SDS JP ja Download
  •  Publications

    How to properly cite this product in your work

    Diagenode strongly recommends using this: Ago (Argonautes) Antibody (Diagenode Cat# C15200167-100 Lot# 003). Click here to copy to clipboard.

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    ACE-score-based Analysis of Temporal miRNA Targetomes During Human Cytomegalovirus Infection Using AGO-CLIP-seq
    Kim S et al.
    Although temporal regulation of gene expression during the course of infection is known to be critical for determining the outcome of host-virus interactions, systematic temporal analysis of the miRNA targetomes during productive viral infection has been technically challenging due to the large range of miRNA-mRNA c...

    Differential RISC association of endogenous human microRNAs predicts their inhibitory potential.
    Flores O, Kennedy EM, Skalsky RL, Cullen BR
    It has previously been assumed that the generally high stability of microRNAs (miRNAs) reflects their tight association with Argonaute (Ago) proteins, essential components of the RNA-induced silencing complex (RISC). However, recent data have suggested that the majority of mature miRNAs are not, in fact, Ago associa...

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