ELAVL3 Antibody, FITC conjugated

What is ELAVL3 and what cellular functions does it perform?

ELAVL3 (ELAV-like protein 3), also known as HuC, is a neuronal-specific RNA-binding protein with a molecular weight of approximately 39.5 kDa. It belongs to the RRM ELAV protein family and binds specifically to AU-rich elements (AREs) in the 3'-UTR of target mRNAs, including VEGF mRNA . ELAVL3 demonstrates brain-specific expression patterns and may be involved in neuronal differentiation and maintenance . The protein contains up to 367 amino acid residues in its canonical form, and up to two different isoforms have been reported .

Functionally, ELAVL3 plays critical roles in:

• Post-transcriptional regulation of gene expression

• Stabilization of specific target mRNAs

• Neuronal differentiation processes

• Maintenance of neuronal phenotype

For experimental detection, researchers should note that while the calculated molecular weight is 38-39 kDa, observed bands in Western blot applications may appear around 37 kDa .

What are the primary applications for FITC-conjugated ELAVL3 antibodies?

FITC-conjugated ELAVL3 antibodies are particularly valuable for direct visualization applications that eliminate the need for secondary antibody incubation steps. The primary applications include:

• Immunofluorescence microscopy: Direct detection of ELAVL3 in fixed tissue sections or cultured cells

• Flow cytometry: Analysis of ELAVL3 expression in cell populations

• Confocal microscopy: High-resolution imaging of ELAVL3 subcellular localization

• Live cell imaging: When using non-fixation compatible formulations

These applications benefit from the direct FITC conjugation, which shows strong signal in neuronal tissues. Verification studies have confirmed successful detection in rat brain, mouse brain, and C6 glioma cell line samples using immunofluorescence techniques .

What tissues and samples are most reliable for ELAVL3 antibody validation?

Based on published validation data, the following samples have been consistently verified for ELAVL3 antibody applications:

When validating a new FITC-conjugated ELAVL3 antibody, these tissue types should be considered as positive controls. Brain tissue is particularly valuable given ELAVL3's neuronal expression pattern . For negative controls, tissues with minimal ELAVL3 expression (such as muscle) can be used to confirm specificity.

How does ELAVL3 contribute to neuroendocrine prostate cancer development?

Recent research has revealed that ELAVL3 plays a crucial role in neuroendocrine prostate cancer (NEPC) through a positive feedback mechanism with MYCN:

• ELAVL3 is specifically upregulated in neuroendocrine prostate cancer

• MYCN transcriptionally upregulates ELAVL3 by binding to consensus sequences (CACGTG) in the ELAVL3 promoter region

• ELAVL3 then binds to and stabilizes MYCN mRNA through interaction with AU-rich elements in the 3'-UTR

• This creates a positive feedback loop that drives neuroendocrine differentiation

• ELAVL3 also stabilizes RICTOR mRNA, which contributes to the activation of the PI3K/AKT/mTOR pathway

Importantly, overexpression of ELAVL3 alone has been shown to be sufficient to induce neuroendocrine phenotype in prostate adenocarcinoma cells . ELAVL3 expression correlates positively with neuroendocrine biomarkers (SYP, CHGA, CHGB) and inversely with androgen receptor-related genes (AR, KLK3, NKX3-1) .

FITC-conjugated ELAVL3 antibodies can be valuable tools for studying these mechanisms through direct visualization of ELAVL3 in prostate cancer tissue samples.

What methodological approaches can optimize FITC-conjugated ELAVL3 antibody signal in multiplexed immunofluorescence studies?

When designing multiplexed immunofluorescence experiments with FITC-conjugated ELAVL3 antibodies, consider these optimization strategies:

• Spectral considerations: FITC emits at approximately 520 nm (green). For multiplexing, select additional fluorophores with minimal spectral overlap, such as:

  • DAPI (blue, nuclear stain)

  • Cy3/TRITC (red)

  • Cy5/APC (far red)

• Signal amplification strategies:

  • Use tyramide signal amplification (TSA) for weak signals

  • Apply suitable antifade mounting media to prevent photobleaching of FITC

  • Consider sequential detection for challenging multiplex panels

• Protocol optimization:

  • Extend incubation times (overnight at 4°C) for deeper tissue penetration

  • Use appropriate diluent buffers (PBS with 0.03% Proclin 300)

  • Test different fixation protocols (4% PFA typically preserves ELAVL3 epitopes)

• Validated co-labeling targets:

  • Neuronal markers (Map2, NeuN) for co-localization studies

  • MYCN for investigating the ELAVL3/MYCN feedback loop

  • Neuroendocrine markers (SYP, CHGA, CHGB) to correlate with ELAVL3 expression

What are the key considerations for validating specificity of FITC-conjugated ELAVL3 antibodies?

Rigorous validation is essential for ensuring reliable results with FITC-conjugated ELAVL3 antibodies:

• Knockout/knockdown controls:

  • Compare staining in ELAVL3 wildtype versus knockout models

  • Use siRNA/shRNA-mediated knockdown of ELAVL3 (as demonstrated in studies with LNCaP/AR/shTP53/shRB1 cells)

  • Analyze both fluorescence imaging and Western blot to confirm specificity

• Peptide competition assays:

  • Pre-incubate antibody with excess immunizing peptide (such as recombinant ELAVL3 protein 1-367aa)

  • Compare signal with and without peptide competition

• Cross-reactivity assessment:

  • Test reactivity against other ELAV family members (ELAVL1, ELAVL2, ELAVL4)

  • Analyze tissues known to express different ELAV proteins

• Application-specific controls:

  • For flow cytometry: Include isotype controls conjugated to FITC

  • For immunofluorescence: Include secondary-only controls and autofluorescence controls

• Correlation with functional readouts:

  • Verify that ELAVL3 detection correlates with expected biological activities, such as binding to target mRNAs or induction of neuroendocrine markers

What is the recommended protocol for using FITC-conjugated ELAVL3 antibodies in immunofluorescence studies?

The following protocol is optimized for FITC-conjugated ELAVL3 antibody use in immunofluorescence:

Materials required:

• FITC-conjugated ELAVL3 antibody

• Blocking solution (5% normal serum in PBS-T)

• PBS (pH 7.4)

• 4% Paraformaldehyde

• 0.1% Triton X-100

• Mounting medium with anti-fade properties

• DAPI (optional for nuclear counterstaining)

Protocol:

• Sample preparation:

  • Fix tissue sections or cultured cells with 4% paraformaldehyde for 15 minutes

  • Wash 3 times with PBS, 5 minutes each

• Permeabilization:

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

  • Wash 3 times with PBS, 5 minutes each

• Blocking:

  • Incubate with blocking solution for 1 hour at room temperature

  • Do not wash after blocking

• Primary antibody incubation:

  • Dilute FITC-conjugated ELAVL3 antibody 1:50-1:200 in blocking solution

  • Incubate overnight at 4°C in a humidified chamber protected from light

  • Wash 3 times with PBS, 5 minutes each

• Counterstaining:

  • Incubate with DAPI solution (1 μg/ml) for 5 minutes

  • Wash 3 times with PBS, 5 minutes each

• Mounting:

  • Mount with anti-fade mounting medium

  • Seal edges with nail polish

• Imaging:

  • Use appropriate filter sets for FITC (excitation: 490 nm, emission: 520 nm)

  • Image within 1-2 weeks for optimal signal

Based on validation studies, this protocol has been successfully used with mouse brain, rat brain, and C6 cell samples .

How should researchers troubleshoot weak or nonspecific signals when using FITC-conjugated ELAVL3 antibodies?

When encountering signal issues with FITC-conjugated ELAVL3 antibodies, consider these troubleshooting approaches:

For weak signals:

• Antibody concentration optimization:

  • Test a concentration series from 1:25 to 1:200

  • Higher concentrations of antibody (1:50) may be required for immunofluorescence compared to standard immunohistochemistry

• Antigen retrieval enhancement:

  • Test citrate buffer (pH 6.0) heat-mediated antigen retrieval

  • Consider proteolytic-induced epitope retrieval with proteinase K

• Signal amplification:

  • Apply tyramide signal amplification (TSA)

  • Use higher-sensitivity detection systems

• Sample preparation improvements:

  • Minimize time between tissue collection and fixation

  • Optimize fixation duration (over-fixation can mask epitopes)

  • For brain tissue, transcardial perfusion fixation may improve results

For nonspecific signals:

• Background reduction:

  • Increase blocking time and concentration (try 10% normal serum)

  • Add 0.1-0.3% Triton X-100 to antibody diluent

  • Include 0.1% BSA in washing buffers

• Antibody specificity verification:

  • Test antibody on known negative control tissues

  • Perform peptide blocking controls

  • Test multiple lots of the antibody if available

• Autofluorescence management:

  • Treat sections with 0.1% Sudan Black B in 70% ethanol

  • Consider using TrueBlack® or similar autofluorescence quenchers

  • Adjust imaging settings to minimize autofluorescence detection

What are the optimal storage conditions for maintaining FITC-conjugated ELAVL3 antibody activity?

To maintain optimal activity of FITC-conjugated ELAVL3 antibodies, follow these storage guidelines:

• Long-term storage:

  • Store at -20°C in the dark

  • Aliquot to avoid repeated freeze-thaw cycles

  • Glycerol-based storage buffer (50% glycerol, 0.01M PBS, pH 7.4) helps prevent freezing damage

  • Use preservative (such as 0.03% Proclin 300) to prevent microbial growth

• Working solution handling:

  • Keep on ice and protected from light during experiments

  • Return to -20°C promptly after use

  • Avoid exposure to strong light sources when not in use

  • Consider adding protein carriers (BSA) to diluted antibody solutions

• Stability considerations:

  • Typically valid for 12 months when properly stored

  • Monitor for precipitation before each use

  • FITC fluorescence may gradually decrease over time due to photobleaching

  • Consider refreshing working stocks every 6 months

• Transportation:

  • Transport on ice packs

  • Ensure protection from light during shipping

  • Allow to equilibrate to room temperature before opening to prevent condensation

Proper storage is critical as FITC conjugates are particularly sensitive to light exposure and protein degradation.

How can FITC-conjugated ELAVL3 antibodies be used to study the ELAVL3/MYCN feedback loop in cancer models?

FITC-conjugated ELAVL3 antibodies can be valuable tools for investigating the ELAVL3/MYCN regulatory axis in cancer through these approaches:

• Co-localization studies:

  • Perform double immunofluorescence with FITC-conjugated ELAVL3 antibodies and MYCN antibodies (different fluorophore)

  • Analyze subcellular co-localization patterns in cancer cell lines and patient-derived xenografts

• Expression correlation analysis:

  • Quantify ELAVL3 signal intensity using FITC-conjugated antibodies across:

    • Treatment conditions (e.g., enzalutamide resistance models)

    • MYCN-overexpression or knockdown models

    • Patient samples with varying MYCN expression levels

• Therapeutic response monitoring:

  • Track ELAVL3 expression changes following treatment with:

    • MYCN pathway inhibitors (e.g., MLN8237)

    • Pyrvinium pamoate (shown to disrupt ELAVL3-MYCN interaction)

    • AR pathway modulators like enzalutamide

• Live cell imaging applications:

  • Monitor real-time changes in ELAVL3 expression following MYCN modulation

  • Track dynamics of ELAVL3 expression during neuroendocrine differentiation

• Flow cytometry analysis:

  • Quantify ELAVL3 expression levels in heterogeneous tumor populations

  • Correlate with neuroendocrine markers in various prostate cancer cell lines

This approach is particularly relevant given that ELAVL3 overexpression has been shown to increase sensitivity to MLN8237, an inhibitor of the MYCN/AURKA pathway (IC₅₀ of 0.067 μM compared with IC₅₀ = 1.084 μM for control cells) , indicating the therapeutic relevance of this feedback loop.

What technical considerations should be addressed when using FITC-conjugated ELAVL3 antibodies in extracellular vesicle research?

Recent research has shown that ELAVL3 can be released in extracellular vesicles and induce neuroendocrine differentiation of adenocarcinoma cells through intercellular mechanisms . When investigating this phenomenon with FITC-conjugated ELAVL3 antibodies:

• Vesicle isolation optimization:

  • Use differential ultracentrifugation or size exclusion chromatography

  • Confirm vesicle isolation with nanoparticle tracking analysis

  • Verify membrane integrity before antibody application

• Permeabilization protocols:

  • For internal EV proteins, apply mild detergents (0.01% saponin)

  • Test different permeabilization conditions to maintain vesicle structure

  • Consider fixation with 2% paraformaldehyde before permeabilization

• Background reduction strategies:

  • Apply extensive washing steps after antibody incubation

  • Use 0.1 μm filtered buffers to reduce background particulates

  • Consider using EV-depleted serum in culture media

• Signal amplification approaches:

  • Consider bead-based flow cytometry for enhanced detection

  • Use fluorescence-triggered high-resolution flow cytometry

  • Apply super-resolution microscopy techniques for detailed imaging

• Controls and validation:

  • Include isotype controls conjugated to FITC

  • Perform antibody titration specifically for EV applications

  • Confirm ELAVL3 presence by orthogonal methods (Western blot)

How can researchers quantitatively assess ELAVL3 expression levels using FITC-conjugated antibodies in patient samples?

For quantitative assessment of ELAVL3 expression in patient samples using FITC-conjugated antibodies:

• Standardized tissue processing:

  • Establish consistent fixation protocols (e.g., 10% neutral buffered formalin for 24h)

  • Process all samples using identical protocols

  • Include calibration standards on each slide

• Image acquisition standardization:

  • Use consistent exposure settings across all samples

  • Acquire images using identical microscope configurations

  • Include fluorescence calibration beads in each imaging session

• Quantification methods:

  • Measure mean fluorescence intensity (MFI) in defined regions of interest

  • Apply automated segmentation algorithms to identify ELAVL3-positive cells

  • Calculate percentage of ELAVL3-positive cells in the total population

• Normalization approaches:

  • Normalize to internal controls (housekeeping proteins)

  • Subtract background fluorescence from each measurement

  • Use ratio metrics (e.g., ELAVL3/DAPI ratio) for comparison across samples

• Correlation with clinical parameters:

  • Compare ELAVL3 expression with prognostic indicators

  • Analyze relationship between ELAVL3 levels and:

    • Neuroendocrine markers (SYP, CHGA, CHGB)

    • MYCN expression levels

    • Treatment response to specific therapies

    • Patient outcomes and survival metrics

This quantitative approach allows researchers to establish clinically relevant cutoff values for ELAVL3 expression that might predict treatment response or disease progression in neuroendocrine prostate cancer.

What are the recommended protocols for chromatin immunoprecipitation (ChIP) assays investigating MYCN binding to the ELAVL3 promoter?

While FITC-conjugated antibodies are not typically used for ChIP, researchers investigating the ELAVL3/MYCN feedback loop may want to complement ELAVL3 immunofluorescence studies with ChIP analysis of MYCN binding to the ELAVL3 promoter:

• Primer design for ELAVL3 promoter:

  • Target the MYCN binding sites (BS1 and BS2) containing the consensus sequence CACGTG

  • Design primers flanking these binding sites for qPCR analysis

  • Include primers for known MYCN target genes as positive controls

• ChIP protocol optimization:

  • Crosslink cells with 1% formaldehyde for 10 minutes

  • Sonicate to generate DNA fragments of 200-500 bp

  • Use anti-MYCN antibodies for immunoprecipitation

  • Include appropriate negative controls (IgG, non-specific regions)

• Data analysis approaches:

  • Calculate fold enrichment relative to IgG control

  • Compare enrichment at BS1 and BS2 sites

  • Assess effects of MYCN modulation on binding efficiency

• Validation strategies:

  • Confirm findings with luciferase reporter assays using ELAVL3 promoter constructs

  • Test mutated constructs where the CACGTG consensus sequence is replaced with CTGCGG

  • Perform CRISPR/Cas9-mediated mutation of endogenous binding sites

This approach has successfully demonstrated direct binding of MYCN to the ELAVL3 promoter in previous studies, particularly at the BS1 site in LNCaP cells .

What experimental designs can evaluate the therapeutic potential of disrupting the ELAVL3/MYCN interaction in cancer models?

Based on recent findings about pyrvinium pamoate's ability to disrupt the ELAVL3/MYCN interaction , researchers can design experiments to evaluate therapeutic potential: