AGO3 Antibody, FITC conjugated

What is AGO3 and what cellular functions does it serve?

AGO3 (Argonaute 3, also known as eIF2C3) is a member of the Argonaute family of proteins involved in RNA interference (RNAi) mediated gene silencing through siRNA and miRNA effectors. It functions as a component of the RNA-Induced Silencing Complex (RISC) and microRNA (miRNA)-containing ribonucleoprotein particles (miRNP). Unlike its family member AGO2, AGO3 lacks endonuclease activity and does not appear to cleave target mRNA molecules. AGO3 is primarily involved in translational repression rather than direct mRNA cleavage. The protein contains both PAZ and PIWI domains, which are characteristic of the Argonaute family . AGO3 is primarily localized in the cytoplasm and participates in the stabilization of small RNA derivatives (riRNA) and subsequent riRNA-dependent degradation of mRNAs by recruiting decapping complexes involving EDC4 .

What is the molecular structure and subcellular localization of AGO3?

AGO3 is a protein with a molecular weight of approximately 97 kDa and consists of 860 amino acids in humans. Its structure includes the characteristic PAZ domain (which binds to the 3' ends of small RNAs) and the PIWI domain (which structurally resembles RNase H). AGO3 is predominantly localized in the cytoplasm , where it associates with processing bodies (P-bodies) that are involved in mRNA degradation and storage. The protein functions by binding to short RNAs such as microRNAs and repressing the translation of complementary mRNAs. AGO3 is encoded by the EIF2C3 gene (Gene ID: 192669) .

How does FITC conjugation affect the functionality of AGO3 antibodies?

FITC (Fluorescein Isothiocyanate) conjugation provides direct fluorescent labeling of the AGO3 antibody, eliminating the need for secondary antibody detection in immunofluorescence applications. The conjugation process attaches the FITC molecule to the antibody while preserving the antibody's ability to recognize and bind to AGO3 protein epitopes. When working with FITC-conjugated antibodies, researchers should be aware that the fluorophore is sensitive to photobleaching and has an excitation maximum at approximately 495 nm and an emission maximum at approximately 519 nm. For optimal results, the FITC-conjugated AGO3 antibody should be stored protected from light at -20°C and aliquoted into multiple vials to avoid repeated freeze-thaw cycles, which can damage both the antibody and the fluorophore .

What are the recommended applications for FITC-conjugated AGO3 antibodies?

FITC-conjugated AGO3 antibodies are particularly useful for fluorescence-based applications. According to product information, the main applications include:

• Immunofluorescence (IF) on paraffin-embedded tissues (IHC-P) with recommended dilutions of 1:50-200

• Immunofluorescence on frozen sections (IHC-F)

• Immunocytochemistry (ICC) for cultured cells

• Western Blotting (WB)

The direct fluorescent labeling makes these antibodies especially valuable for multi-color immunofluorescence staining, co-localization studies, and flow cytometry applications. When designing experiments, researchers should consider the specific reactivity of the antibody, which typically includes human, mouse, and rat samples, with some products showing broader cross-reactivity with other species such as dog, cow, pig, horse, chicken, and zebrafish .

What are the optimal protocols for immunofluorescence using FITC-conjugated AGO3 antibodies?

For optimal immunofluorescence results with FITC-conjugated AGO3 antibodies, follow this methodology:

For paraffin-embedded tissue sections (IHC-P):

• Deparaffinize and rehydrate sections through a graded ethanol series

• Perform antigen retrieval (typically heat-induced epitope retrieval in citrate buffer pH 6.0)

• Block endogenous peroxidase activity with 3% H₂O₂ (if applicable)

• Block non-specific binding with 5-10% normal serum in PBS for 1 hour

• Incubate with FITC-conjugated AGO3 antibody at 1:50-200 dilution in blocking buffer overnight at 4°C

• Wash thoroughly with PBS (3 × 5 minutes)

• Counterstain nuclei with DAPI

• Mount with anti-fade mounting medium

• Visualize using a fluorescence microscope with appropriate filter sets (excitation ~495 nm, emission ~519 nm)

For cultured cells (ICC):

• Fix cells with 4% paraformaldehyde for 15 minutes

• Permeabilize with 0.1-0.25% Triton X-100 in PBS for 10 minutes

• Block with 1-5% BSA or 10% normal serum in PBS for 1 hour

• Incubate with FITC-conjugated AGO3 antibody at appropriate dilution in blocking buffer for 1-2 hours at room temperature or overnight at 4°C

• Wash 3 × 5 minutes with PBS

• Counterstain nuclei with DAPI

• Mount and visualize

Store the antibody at -20°C and protect from light to maintain fluorescence intensity. Aliquot the antibody to avoid repeated freeze-thaw cycles .

How can I validate the specificity of FITC-conjugated AGO3 antibodies?

To validate the specificity of FITC-conjugated AGO3 antibodies, implement these methodological approaches:

• Positive and negative controls:

  • Use cell lines or tissues known to express AGO3 as positive controls

  • Use AGO3 knockout cells or tissues as negative controls

  • Compare with known expression patterns from literature or databases

• Peptide competition assay:

  • Pre-incubate the antibody with the immunizing peptide before application

  • If the antibody is specific, the signal should be significantly reduced or eliminated

• Cross-validation with different antibodies:

  • Compare staining patterns with other validated AGO3 antibodies targeting different epitopes

  • Consider using non-conjugated primary antibodies with secondary detection methods as reference

• Western blot verification:

  • Confirm that the antibody recognizes a protein of the expected size (~97 kDa for AGO3)

  • Look for a single specific band at the correct molecular weight

• siRNA knockdown:

  • Perform RNA interference to reduce AGO3 expression

  • Verify reduced signal intensity in knockdown samples compared to controls

Remember that the FITC-conjugated Argonaute 3/eIF2C3 polyclonal antibody is derived from a KLH-conjugated synthetic peptide corresponding to amino acid region 251-350 of the 860 amino acid human AGO3 protein , so expression patterns should match this epitope specificity.

What are common issues with FITC-conjugated AGO3 antibodies and how can they be resolved?

When troubleshooting, remember that AGO3 is primarily cytoplasmic , so nuclear staining might indicate non-specific binding. Additionally, ensure that imaging is performed with appropriate filter sets for FITC (excitation ~495 nm, emission ~519 nm).

How can I differentiate between AGO3 and other Argonaute family proteins in my experiments?

Differentiating between AGO3 and other Argonaute family proteins (AGO1, AGO2, AGO4) requires careful experimental design:

• Antibody selection: Choose antibodies specifically targeting unique regions of AGO3 not conserved in other Argonaute proteins. The epitope information is crucial - the polyclonal antibody described in the search results targets amino acids 251-350 of human AGO3 .

• Co-localization studies: Perform dual immunofluorescence with antibodies against different Argonaute proteins labeled with distinct fluorophores to observe differential localization patterns.

• Functional assays: AGO3 lacks endonuclease activity, unlike AGO2 . In functional assays, AGO3 will not cleave perfectly complementary targets, whereas AGO2 will. This functional difference can help distinguish between these proteins.

• Expression analysis: Use quantitative PCR to determine relative expression levels of different Argonaute transcripts in your experimental system.

• RNAi approaches: Perform selective knockdown of AGO3 using siRNAs that specifically target AGO3 but not other family members, then confirm specificity with the antibody.

• Immunoprecipitation followed by mass spectrometry: This approach can definitively identify the protein being recognized by the antibody based on peptide sequences.

• Binding partners: AGO3 interacts with specific miRNAs and mRNAs that may differ from those interacting with other Argonaute proteins .

When interpreting results, note that AGO3 primarily functions in translational repression rather than mRNA cleavage, which is the main function of AGO2 .

How should I interpret AGO3 subcellular localization patterns?

When interpreting AGO3 subcellular localization patterns using FITC-conjugated antibodies, consider these key points:

• Normal distribution: AGO3 is primarily localized in the cytoplasm , often concentrated in processing bodies (P-bodies) that appear as cytoplasmic foci. Any substantial nuclear staining should be carefully validated.

• P-body association: AGO3 typically co-localizes with other P-body markers (like GW182, DCP1, DCP2). Consider co-staining experiments to confirm P-body localization.

• Stress conditions: Under cellular stress (oxidative stress, heat shock, etc.), AGO3 distribution may change, potentially showing increased accumulation in stress granules. Compare stressed vs. unstressed cells to document these changes.

• Cell cycle dependence: AGO3 distribution may vary throughout the cell cycle. Note cell morphology and potentially co-stain with cell cycle markers to correlate localization patterns with cell cycle phases.

• Experimental artifacts: Overexpression of AGO3 can lead to artifactual localization patterns. Compare endogenous staining with overexpression systems carefully.

• Fixation artifacts: Different fixation methods can affect AGO3 localization patterns. Cross-validate with multiple fixation protocols.

• Comparison with functional data: Correlate localization patterns with functional readouts of miRNA activity to establish biological relevance.

In disease states or experimental manipulations, changes in AGO3 localization (such as nuclear accumulation or dispersion from P-bodies) may indicate alterations in miRNA pathway functionality that correlate with changes in target gene expression .

How can FITC-conjugated AGO3 antibodies be used in RNA-protein interaction studies?

FITC-conjugated AGO3 antibodies offer powerful approaches for investigating RNA-protein interactions:

• Immunofluorescence-FISH (IF-FISH) co-localization:

  • Combine FITC-AGO3 immunofluorescence with fluorescence in situ hybridization (FISH) for specific miRNAs or target mRNAs

  • This allows visualization of spatial relationships between AGO3 and its RNA partners

  • Use spectral unmixing if needed to distinguish FITC signal from other fluorophores

• Proximity Ligation Assay (PLA) with RNA modification:

  • Adapt standard PLA protocols to detect AGO3-RNA interactions

  • Use FITC-conjugated AGO3 antibody with a complementary antibody against RNA-binding proteins

  • Alternatively, use biotinylated RNA probes with streptavidin-conjugated PLA probes

• Live-cell imaging with complementary techniques:

  • While FITC-conjugated antibodies can't be used directly for live cells, they can validate findings from live-cell systems

  • Correlate fixed-cell FITC-AGO3 staining with data from live-cell experiments using fluorescently tagged AGO3

• FRET-based approaches:

  • Use FITC as a donor fluorophore in Förster Resonance Energy Transfer (FRET) studies

  • Pair with acceptor fluorophores conjugated to antibodies against RNA-binding proteins or in situ hybridization probes

• Immuno-electron microscopy:

  • Convert FITC signals to electron-dense materials for ultrastructural visualization

  • Map AGO3-RNA interactions at nanometer resolution

These advanced techniques can reveal mechanistic insights into how AGO3 participates in RNA silencing through interaction with miRNAs and target mRNAs, particularly its role in translational repression independent of endonucleolytic cleavage .

What is the role of AGO3 in disease pathways and how can FITC-conjugated antibodies help in investigating these mechanisms?

AGO3 has been implicated in various disease mechanisms, and FITC-conjugated antibodies offer valuable tools for investigation:

• Cancer biology:

  • Altered AGO3 expression and localization have been observed in various cancers

  • FITC-conjugated AGO3 antibodies can be used to:

    • Quantify expression levels in tumor vs. normal tissues

    • Track subcellular redistributions associated with malignant transformation

    • Correlate with miRNA dysregulation patterns specific to cancer types

• Neurodegenerative diseases:

  • miRNA dysregulation is implicated in conditions like Alzheimer's and Parkinson's

  • AGO3-FITC immunostaining can:

    • Reveal altered distribution in affected neurons

    • Detect co-localization with disease-specific protein aggregates

    • Monitor stress granule associations in neurodegeneration models

• Viral infections:

  • Some viruses manipulate host miRNA machinery for replication

  • FITC-AGO3 antibodies enable:

    • Visualization of AGO3 redistribution during viral infection

    • Co-localization studies with viral components

    • Assessment of virus-induced alterations in miRNA-mediated silencing

• Inflammatory disorders:

  • AGO3 may regulate inflammatory gene expression through miRNAs

  • Fluorescence-based quantification can:

    • Measure changes in AGO3 levels during inflammation

    • Track AGO3 in immune cell activation and differentiation

• Developmental disorders:

  • AGO3's role in stabilizing small RNA derivatives (riRNA) and mRNA decapping may impact developmental gene regulation

  • FITC-conjugated antibodies permit:

    • Tissue-specific expression analysis during development

    • Correlation of spatial expression patterns with developmental abnormalities

When investigating disease mechanisms, combining FITC-AGO3 immunostaining with patient-derived samples and disease models can provide insights into how alterations in AGO3 function contribute to pathogenesis and potential therapeutic targets in the RNA interference pathway.

How can FITC-conjugated AGO3 antibodies be used in combination with other molecular techniques for comprehensive research?

Integrating FITC-conjugated AGO3 antibodies with complementary molecular techniques creates powerful research paradigms:

• Integration with sequencing technologies:

  • Fluorescence-activated cell sorting (FACS) of AGO3-FITC positive cells followed by RNA-seq or small RNA-seq

  • Correlation of AGO3 expression/localization patterns with transcriptome profiles

  • CLIP-seq validation using AGO3 antibodies to confirm binding partners identified in imaging

• Combination with live-cell dynamics:

  • Fixed-cell FITC-AGO3 imaging to validate GFP-AGO3 live-cell experiments

  • Pulse-chase experiments to track AGO3 dynamics over time

  • Photoconversion studies paired with subsequent FITC-AGO3 immunofluorescence

• Multi-omics approaches:

  • AGO3-FITC immunostaining of cells prepared for spatial transcriptomics

  • Integration of proteomics data with AGO3 localization patterns

  • Correlation of AGO3 distribution with epigenomic features

• High-throughput screening applications:

  • FITC-AGO3 immunofluorescence in cell-based screens for miRNA pathway modulators

  • Automated image analysis of AGO3 localization changes in response to compound libraries

  • Validation of screen hits with biochemical assays using non-conjugated AGO3 antibodies

• Super-resolution microscopy:

  • FITC-AGO3 antibodies can be used with techniques like STORM or STED

  • Nanoscale visualization of AGO3 within P-bodies and other subcellular structures

  • Co-localization with RNA targets at molecular resolution

• In vivo applications:

  • Ex vivo staining of tissue sections from experimental animals

  • Correlation with in vivo functional studies of miRNA activity

  • Analysis of AGO3 expression in animal models of disease

This multidisciplinary approach leverages the direct visualization capabilities of FITC-AGO3 antibodies while enriching the findings with complementary molecular data, providing a comprehensive understanding of AGO3's roles in RNA silencing complexes and disease mechanisms .

What controls should be included when using FITC-conjugated AGO3 antibodies in experimental designs?

A robust experimental design with FITC-conjugated AGO3 antibodies requires these essential controls:

• Antibody specificity controls:

  • Peptide competition/blocking: Pre-incubate antibody with immunizing peptide to block specific binding

  • Knockout/knockdown validation: Use AGO3 knockout or siRNA-depleted samples as negative controls

  • Isotype control: Include FITC-conjugated isotype-matched non-specific antibody (e.g., rabbit IgG-FITC) to assess non-specific binding

• Fluorescence controls:

  • Autofluorescence control: Unstained samples to determine background autofluorescence

  • Single-color controls: When performing multi-color experiments, include single-stained samples for each fluorophore

  • Photobleaching control: Monitor signal stability over multiple exposures

• Technical controls:

  • Secondary antibody-only control: If using additional layers of detection

  • Fixation control: Compare different fixation methods to identify potential artifacts

  • Blocking optimization: Samples with different blocking conditions to minimize background

• Biological controls:

  • Positive control tissue/cells: Samples known to express AGO3 (based on literature)

  • Related protein controls: Include staining for other Argonaute family members (AGO1, AGO2, AGO4) to assess specificity

  • Treatment conditions: Include relevant positive and negative treatment controls

• Validation with orthogonal methods:

  • Western blot correlation: Verify that IF signal intensity correlates with protein levels detected by Western blot

  • qPCR validation: Confirm that protein expression patterns correlate with mRNA expression

A well-designed experiment should include comprehensive documentation of antibody characteristics: host species (rabbit), clonality (polyclonal), immunogen details (synthetic peptide corresponding to amino acids 251-350/860 of human AGO3), and storage conditions (-20°C, protected from light) .

How can I optimize FITC-conjugated AGO3 antibody performance for different tissue types?

Optimizing FITC-conjugated AGO3 antibody performance across tissue types requires systematic adjustment of multiple parameters:

Additional optimization strategies:

• Tissue-specific blocking:

  • Brain: Add 0.1-0.3% Triton X-100 in blocking solution

  • Liver/kidney: Include 0.1% Sudan Black to reduce autofluorescence

  • High-lipid tissues: Include 0.05% saponin in blocking buffer

• Signal amplification options:

  • For tissues with low AGO3 expression, consider using a non-conjugated primary antibody with a FITC-conjugated secondary antibody for signal amplification

  • Tyramide signal amplification can be used for very low abundance targets

• Iterative optimization:

  • For each new tissue type, prepare a titration series of antibody dilutions (1:25, 1:50, 1:100, 1:200, 1:400)

  • Test multiple antigen retrieval methods side-by-side

  • Document staining patterns carefully with standardized exposure settings

When transitioning between different tissue types, maintain detailed records of optimization parameters to build a reference database for future experiments.

What are the critical factors affecting FITC fluorescence stability and how can they be managed?

Maintaining optimal FITC fluorescence stability requires careful management of several critical factors:

• Light exposure:

  • FITC is highly susceptible to photobleaching

  • Prevention strategies:

    • Store antibody solutions in amber vials or wrapped in aluminum foil

    • Work under reduced lighting conditions

    • Minimize exposure during microscopy by using neutral density filters

    • Use minimal exposure times during image acquisition

• pH sensitivity:

  • FITC fluorescence is optimal at pH 7.5-8.0 and diminishes significantly below pH 7.0

  • Management approaches:

    • Use buffers with consistent pH and sufficient buffering capacity

    • Monitor pH of solutions, especially during long-term storage

    • Consider pH-insensitive alternatives (like Alexa Fluor 488) for acidic environments

• Storage conditions:

  • Proper storage is critical for maintaining antibody integrity and fluorescence

  • Best practices:

    • Store at -20°C as recommended

    • Aliquot the antibody upon receipt to avoid repeated freeze-thaw cycles

    • Add protein stabilizers (like 1% BSA) to diluted antibody solutions

    • Never store diluted antibody solutions for extended periods

• Mounting media selection:

  • Mounting media significantly impact fluorescence longevity

  • Recommendations:

    • Use anti-fade mounting media containing anti-photobleaching agents

    • Consider media with hardening properties for long-term storage

    • Seal edges of coverslips with nail polish for additional protection

• Chemical environment:

  • Certain chemicals can quench FITC fluorescence

  • Considerations:

    • Avoid high concentrations of halogens or transition metals in buffers

    • Minimize exposure to oxidizing agents

    • Be cautious with fixatives containing high aldehyde concentrations

• Image acquisition strategies:

  • Smart imaging approaches can preserve signal

  • Techniques:

    • Focus using differential interference contrast (DIC) before switching to fluorescence

    • Use computational approaches (like deconvolution) to enhance low-intensity signals

    • Consider spectral unmixing to separate FITC signal from autofluorescence

• Documentation and quantification:

  • Include fluorescence intensity standards in experiments

  • Image all experimental groups in a single session with identical settings

  • Use automated exposure control where possible to standardize imaging