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, 317 (1-2), 38-44

Detection of the Argonaute Protein Ago2 and microRNAs in the RNA Induced Silencing Complex (RISC) Using a Monoclonal Antibody

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Detection of the Argonaute Protein Ago2 and microRNAs in the RNA Induced Silencing Complex (RISC) Using a Monoclonal Antibody

Keigo Ikeda et al. J Immunol Methods.

Abstract

MicroRNAs (miRNAs) are short RNA molecules responsible for post-transcriptional gene silencing by the degradation or translational inhibition of their target messenger RNAs (mRNAs). This process of gene silencing, known as RNA interference (RNAi), is mediated by highly conserved Argonaute (Ago) proteins which are the key components of the RNA induced silencing complex (RISC). In humans, Ago2 is responsible for the endonuclease cleavage of targeted mRNA and it interacts with the mRNA-binding protein GW182, which is a marker for cytoplasmic foci referred to as GW bodies (GWBs). We demonstrated that the anti-Ago2 monoclonal antibody 4F9 recognized GWBs in a cell cycle dependent manner and was capable of capturing miRNAs associated with Ago2. Since Ago2 protein is the effector protein of RNAi, anti-Ago2 monoclonal antibody may be useful in capturing functional miRNAs.

Figures

Fig. 1
Fig. 1
Development of mouse monoclonal anti-Ago2 antibody 4F9. (A) ELISA reactivity of 4F9 with purified soluble recombinant Ago2 protein. (B) Representative immunoprecipitation analysis of 4F9 reactivity using K562 cell extracts (K562) and in vitro translation product of Ago2 recombinant protein (Ago) both labeled with [35S]-methionine. Immune mouse anti-Ago2 serum (IMS) is used for positive control and normal mouse serum (NMS) as negative control.
Fig. 2
Fig. 2
Immunofluorescence analysis of 4F9.(A) Monoclonal 4F9 (green) recognized cytoplasmic foci identified as GW bodies (GWBs) by costaining with a human index serum known to contain antibodies to anti-Ago2 and GW182 (red). HeLa cells (left panels) were fixed as described in Methods. HEp-2 cells (right panels) were on commercially prepared slides (ImmunoConcepts Inc). Arrowheads show representative GWBs.(B) Monoclonal 4F9 showed staining of GWBs in a cell cycle dependent manner. HEp-2 cells were costained with 4F9 (green foci, bottom panels) and rabbit anti-phosphohistone H3 serum (red, top panels).
Fig. 3
Fig. 3
Monoclonal 4F9 crossreacted with Ago1, Ago3 and Ago4.Immunoprecipitation analysis of 4F9 was performed using in vitro translation products of Ago1, 3 and 4 recombinant proteins labeled with [35S]-methionine. IMS and mouse anti-golgin-97 antibody were used as positive and negative control, respectively.
Fig. 4
Fig. 4
Argonaute monoclonal antibody captured microRNAs.MicroRNAs were detected in the immunoprecipitates of 4F9 and IMS using RT-PCR. Immunoprecipitates of an unrelated mouse monoclonal anti-golgin-97 were analyzed as a negative control. Total RNAs isolated from human heart were used for a positive control for the RT-PCR.

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