AGO2 Recombinant Monoclonal Antibody

What is AGO2 and why is it significant in molecular biology research?

AGO2 (Argonaute 2) is a protein broadly expressed in somatic cells that associates with microRNAs (miRNAs) and functions as a key actor in RNA silencing pathways. Unlike other Argonaute family members, AGO2 uniquely possesses endonucleolytic or "Slicer" activity, allowing it to execute miRNA-directed cleavage of target mRNAs when the base-pairing between the AGO2-associated miRNA and the mRNA sequence is perfect . As a core component of the RNA-induced silencing complex (RISC), AGO2 plays non-redundant roles in small RNA-guided gene silencing processes, including RNA interference, translation repression, and heterochromatinization . Furthermore, AGO2 has been demonstrated to be essential for embryonic development and functions as a key regulator of B-lymphoid and erythroid development .

How do recombinant monoclonal antibodies against AGO2 differ from conventional antibodies?

Recombinant monoclonal antibodies against AGO2 are produced using in vitro expression systems rather than traditional hybridoma techniques. These systems are developed by cloning specific antibody DNA sequences from immunoreactive rabbits, followed by screening individual clones to select optimal candidates for production . This approach offers several significant advantages over conventional antibodies:

• Enhanced specificity and sensitivity toward the AGO2 target

• Consistent performance between production lots, reducing experimental variability

• Animal origin-free formulations, minimizing ethical concerns

• Broader immunoreactivity to diverse targets due to the larger rabbit immune repertoire

These characteristics make recombinant antibodies particularly valuable for studying complex proteins like AGO2 that require high specificity and reproducibility in experimental settings.

What are the validated applications for AGO2 recombinant monoclonal antibodies?

AGO2 recombinant monoclonal antibodies have been validated for multiple experimental applications through rigorous quality control testing. These applications include:

Note: Optimal working dilutions must be determined by end-users as specimens and experimental conditions may vary .

How can researchers effectively use AGO2 antibodies for studying miRNA-associated mechanisms?

AGO2 monoclonal antibodies have become valuable tools for investigating miRNA-mediated gene regulation. The 4F9 anti-AGO2 monoclonal antibody, for example, has been demonstrated to effectively capture Ago-associated miRNAs in mammalian cells . To study miRNA-associated mechanisms:

• Immunoprecipitation of AGO2-miRNA complexes:

  • Crosslink RNA-protein complexes using formaldehyde or UV irradiation

  • Lyse cells under non-denaturing conditions (typically RIPA buffer supplemented with RNase inhibitors)

  • Incubate lysates with AGO2 antibody (such as 4F9 clone) coupled to protein G beads

  • Wash extensively to remove non-specific interactions

  • Elute bound complexes and analyze associated miRNAs by RT-qPCR or sequencing

• Visualization of AGO2-containing structures:

  • AGO2 antibodies can recognize GW bodies (GWBs) which are cytoplasmic foci serving as sites for mRNA storage and degradation

  • The detection pattern may be cell cycle-dependent

  • Use fluorescently labeled secondary antibodies for co-localization studies with other markers such as GW182

What considerations are important when immunoprecipitating AGO2 for RNA analysis?

Several critical factors must be considered when using AGO2 antibodies for RNA immunoprecipitation:

• RNase-free conditions: All buffers and reagents must be prepared under RNase-free conditions to preserve RNA integrity.

• Antibody specificity: Ensure the selected antibody doesn't cross-react with other Argonaute family members. For instance, the 4F9 monoclonal antibody shows specificity for AGO2 in immunoprecipitation assays .

• Interaction stability: The AGO2-RNA interaction remains stable even under highly stringent conditions (1M NaCl), allowing for rigorous washing steps to eliminate non-specific binding .

• RNA-independent binding verification: The interaction between certain antibodies and AGO2 is maintained even in the presence of RNase, confirming that the antibody recognition is independent of AGO2's RNA binding .

• Control immunoprecipitations: Always include isotype controls and, when possible, AGO2-depleted samples as negative controls.

How can researchers investigate AGO2-protein interactions using recombinant monoclonal antibodies?

AGO2 engages in protein-protein interactions that are critical for its function. Recent research has uncovered important interactions, such as between AGO2 and RAS proteins . To investigate such interactions:

• Co-immunoprecipitation approaches:

  • Use anti-AGO2 antibodies to pull down AGO2 complexes

  • Analyze co-precipitated proteins by mass spectrometry or western blotting

  • Verify interactions under varying stringency conditions (the RAS-AGO2 interaction, for example, remains stable even under 1M NaCl conditions)

• Recombinant protein interaction studies:

  • Purify recombinant AGO2 and potential interacting proteins

  • Perform in vitro binding assays followed by immunoprecipitation with AGO2 antibodies

  • For example, concentration-dependent direct interactions between recombinant AGO2 and both wild-type and mutant KRAS proteins have been demonstrated using this approach

• Domain mapping:

  • Using deletion mutants of AGO2, researchers identified that residues K112 and E114 in AGO2 are critical for a direct association with RAS proteins

  • Similar approaches can be used to map other protein interactions

What methodologies are effective for studying AGO2's role in cancer using recombinant monoclonal antibodies?

AGO2 overexpression has been correlated with several aspects of cancer, including tumor cell growth and patient survival . To investigate AGO2's role in cancer:

• Tissue microarray analysis:

  • Use anti-AGO2 antibodies for immunohistochemical staining of cancer tissue microarrays

  • Compare AGO2 expression levels between tumor and adjacent normal tissues

  • Correlate expression with clinical outcomes and pathological features

• Functional studies in cancer cell lines:

  • Knockdown or overexpress AGO2 in cancer cell lines

  • Use AGO2 antibodies to confirm altered expression levels

  • Assess effects on cell proliferation, migration, invasion, and drug sensitivity

  • For example, AGO2 knockdown has been shown to attenuate cell proliferation in mutant KRAS-expressing cells

• Analysis of AGO2-miRNA interactions in cancer contexts:

  • Immunoprecipitate AGO2 from cancer cells using specific antibodies

  • Profile associated miRNAs and compare between normal and cancer states

  • Identify cancer-specific miRNA-AGO2 interactions that may contribute to disease progression

How can researchers distinguish between different functional states of AGO2?

AGO2 exists in different functional states depending on its interactions and modifications. Researchers can investigate these states using:

• Phosphorylation state-specific detection:

  • Use antibodies that recognize specific phosphorylated residues of AGO2

  • Compare with total AGO2 levels detected by non-phospho-specific antibodies

  • Analyze how phosphorylation affects AGO2's interactions with miRNAs and target mRNAs

• Subcellular localization analysis:

  • AGO2 and associated proteins such as GW182 localize to cytoplasmic GW bodies

  • Immunofluorescence with AGO2 antibodies can reveal changes in localization patterns

  • The formation of GWBs has been linked to miRNA genesis, with inhibition of miRNA maturation leading to GWB disassembly

• Analysis of AGO2-RISC complex composition:

  • Immunoprecipitate AGO2 under different cellular conditions

  • Analyze associated proteins and RNAs to determine complex composition

  • Compare complexes between different cellular contexts (e.g., stress conditions, differentiation states)

What are common challenges in AGO2 antibody-based experiments and how can they be addressed?

Researchers working with AGO2 antibodies may encounter several technical challenges:

• Cross-reactivity with other Argonaute family members:

  • Validate antibody specificity using cells with AGO2 knockdown or knockout

  • Select antibodies targeting regions unique to AGO2, such as those targeting the N-terminal domain that contains 10 residues unique to AGO2 compared to other Argonaute proteins

• Variable expression levels across cell types:

  • Optimize protein loading for western blotting based on known AGO2 expression levels

  • Use positive control cell lines with established AGO2 expression (e.g., HeLa cells)

• Preserving AGO2-RNA interactions:

  • Include RNase inhibitors in lysis buffers when studying AGO2-RNA complexes

  • Consider crosslinking approaches for stabilizing transient interactions

• Antibody batch variation:

  • Use recombinant monoclonal antibodies which offer lot-to-lot consistency

  • Include internal controls to normalize between experiments with different antibody batches

What controls are essential when using AGO2 antibodies in research applications?

Proper experimental controls are critical for ensuring reliable results with AGO2 antibodies:

How should researchers optimize protocols for different applications of AGO2 antibodies?

Each application requires specific optimization approaches:

• Western Blotting:

  • Optimal dilution: Start with 1:1,000 as recommended for most AGO2 antibodies

  • Blocking: 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • Primary antibody incubation: Overnight at 4°C for optimal signal-to-noise ratio

  • Detection system: HRP-conjugated secondary antibodies with ECL detection typically provide sufficient sensitivity

• Immunohistochemistry:

  • Antigen retrieval: Critical for formalin-fixed paraffin-embedded tissues

  • Antibody dilution: Begin with 1:100 for paraffin sections

  • Incubation time: Typically 1-2 hours at room temperature or overnight at 4°C

  • Detection: Polymer-HRP systems often provide better sensitivity than traditional ABC methods

• Immunoprecipitation:

  • Lysis conditions: Non-denaturing buffers preserve protein-protein and protein-RNA interactions

  • Antibody amount: Typically 1-5 μg per sample, depending on target abundance

  • Pre-clearing: Reduce non-specific binding by pre-clearing lysates with protein A/G beads

  • Washing stringency: Multiple washes with increasing salt concentration to remove non-specific binders

How can new AGO2 antibody developments advance understanding of non-canonical AGO2 functions?

Recent research has uncovered several non-canonical functions of AGO2 beyond classical miRNA-mediated gene silencing:

• Dicer-independent miRNA processing:

  • AGO2 can act as an RNA slicer in a Dicer-independent manner and regulate miRNA maturation

  • Antibodies specifically recognizing AGO2-miRNA complexes could help elucidate this pathway

• Transcriptional regulation:

  • AGO2 has been implicated in transcriptional gene silencing through heterochromatinization

  • Chromatin immunoprecipitation using AGO2 antibodies can identify genomic loci where AGO2 participates in transcriptional regulation

• Interaction with oncogenic pathways:

  • AGO2 interaction with RAS proteins enhances cellular transformation

  • AGO2 antibodies can be instrumental in mapping interaction networks with oncoproteins

What methodological advances are improving the utility of AGO2 antibodies in research?

Several technological improvements are enhancing AGO2 antibody applications:

• Recombinant antibody engineering:

  • Expression systems developed by cloning specific antibody DNA sequences from immunoreactive rabbits

  • Screening individual clones for optimal binding characteristics

  • Production of animal origin-free formulations

• Epitope mapping and optimization:

  • Targeting of specific epitopes, such as the N-terminal region of AGO2

  • Development of antibodies that can distinguish between different conformational states of AGO2

• Integration with advanced imaging techniques:

  • Super-resolution microscopy combined with AGO2 antibodies to visualize subcellular localization at nanometer resolution

  • Live-cell imaging using fluorescently tagged nanobodies derived from conventional AGO2 antibodies

How can AGO2 antibodies contribute to translational research and potential therapeutic applications?

AGO2 antibodies are becoming increasingly valuable for translational research:

• Biomarker development:

  • AGO2 overexpression correlates with aspects of cancer including tumor cell growth and patient survival

  • AGO2 antibodies can be used to evaluate AGO2 as a prognostic or predictive biomarker in patient samples

• Target validation:

  • For therapeutic approaches targeting the AGO2-RAS interaction

  • AGO2 knockdown attenuates cell proliferation in mutant KRAS-expressing cells, suggesting therapeutic potential

• Drug discovery:

  • Screening compounds that modulate AGO2 activity or interactions

  • Evaluating effects of candidate drugs on AGO2 localization, complex formation, and function

• Understanding disease mechanisms:

  • AGO2 is essential for embryonic development and regulates B-lymphoid and erythroid development

  • AGO2 antibodies can help elucidate mechanisms of developmental and hematological disorders