AGO3 Antibody, Biotin conjugated
Description
Introduction to AGO3 Antibody, Biotin Conjugated
The AGO3 antibody, biotin conjugated, is a specialized immunological reagent designed for detecting the Argonaute 3 (AGO3) protein, a critical component of RNA-induced silencing complexes (RISCs) involved in post-transcriptional gene regulation. Biotin conjugation enhances the antibody’s utility by enabling high-affinity binding to streptavidin/avidin, facilitating signal amplification and versatile detection methods in assays like ELISA, immunoprecipitation, and flow cytometry .
Structure and Functional Mechanism
Biotinylated AGO3 antibodies consist of:
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Antigen-specific region: Targets AGO3, a 87 kDa protein (UniProt: Q9H9G7) that binds small RNAs (e.g., siRNA, miRNA) to mediate RNA interference .
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Biotin conjugation: Covalently attached biotin molecules enable binding to streptavidin, which is often linked to enzymes (e.g., HRP, alkaline phosphatase) or fluorescent dyes for detection .
| Component | Role |
|---|---|
| AGO3-binding domain | Recognizes AGO3 epitopes (e.g., recombinant full-length AGO3 protein) |
| Biotin tag | Facilitates detection via streptavidin-linked reporters |
Applications in Research
Biotin-conjugated AGO3 antibodies are employed in:
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ELISA: Quantitative analysis of AGO3 levels in lysates or serum .
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Immunoprecipitation (IP): Isolation of AGO3-associated RNAs or proteins (e.g., decapping complexes) .
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Flow Cytometry: Detection of AGO3 in intracellular compartments or on cell surfaces .
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Immunofluorescence: Localization of AGO3 in fixed cells or tissues .
Example Protocol:
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ELISA: Coat plates with AGO3 antigen. Detect bound biotinylated antibody using streptavidin-HRP and chromogenic substrate .
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IP: Use streptavidin-coated beads to pull down AGO3 complexes, followed by RNA/protein analysis .
Role in RNA Regulation
AGO3 stabilizes Alu RNAs and facilitates mRNA degradation:
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Alu RNA stabilization: AGO3 interacts with Alu retrotransposons (e.g., DR2 Alu) to protect them from degradation, enabling their regulatory roles in gene expression .
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mRNA decapping: AGO3 associates with the decapping complex (e.g., EDC4, DCP2) to promote 5’ cap removal and exonuclease-mediated mRNA degradation .
Method: Biotin-labeled RNAs immunoprecipitated with AGO3-specific antibodies confirmed RNA binding and stability .
AGO3 in Retinoic Acid Signaling
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Retinoic acid (atRA) downregulates target mRNAs (e.g., Nanog, Phlda1) via AGO3-dependent mechanisms, as shown by siRNA knockdown experiments .
Challenges and Considerations
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Cross-reactivity: Ensure antibodies are cross-adsorbed to minimize non-specific binding (e.g., human vs. mouse reactivity) .
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Protocol Complexity: Biotin-streptavidin systems require additional steps (e.g., streptavidin-HRP incubation) compared to directly conjugated enzymes .
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Cost: Higher expenses due to biotin conjugation and streptavidin reagents .
Product Specs
**Constituents:** 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Research demonstrates that recombinant AGO3 loaded with miR-20a cleaves complementary target RNAs. PMID: 29040713
- A study unveils the first instance of a vertebrate protein factor, Argonaute-3, specifically influencing the guide-to-passenger-strand ratio of the miRNA let-7a. A multifaceted mechanism for the observed impact of Ago3 on the let-7a-3p passenger strand expression and activity is proposed. PMID: 24100239
- The N-terminal domain of Ago3 inhibits cleavage activity. Amino acids 1-64 exhibit a largely unstructured conformation and form a substantial loop surrounding an area containing two helices and another unstructured loop. PMID: 23665583
- The Ago3 PIWI domain possesses slicing competence, but our Ago3 model indicates multiple interactions between residues within the PIWI and N domains. These interactions may enhance protein rigidity or contribute to other properties that explain Ago3's observed slicing deficiency. PMID: 23748378
- DICER- and AGO3-dependent generation of retinoic acid-induced DR2 Alu RNAs regulates human stem cell proliferation. PMID: 23064648
- Ago3 exhibits the capacity to efficiently load microRNAs in the absence of Ago1 and Ago2, despite a significant reduction in global microRNA expression. PMID: 22474261
- Reliable predictions of miRNA affinity to Ago2 AND Ago3 proteins were made. PMID: 21634124
- The specificity of RNA interference is dependent on the relative concentrations of Ago1, Ago3, and Ago4 compared to Ago2. PMID: 18771919
- EIF@C3 protein is expressed in both Schwann and neuron-type differentiating cells. PMID: 19393748
