ELAVL3 Antibody, Biotin conjugated

What is ELAVL3 and why is it important in research?

ELAVL3, also known as HUC or HUCL, is a member of the RNA-binding protein family with brain-specific expression. This 39.5 kDa protein (367 amino acids in its canonical form) binds to AU-rich element (ARE) sequences in target mRNAs . ELAVL3 has gained research significance due to its critical role in:

• Neural development and function as a neuron-specific marker

• Post-transcriptional regulation of gene expression

• Potential involvement in neurodegenerative conditions

• Emerging role as a driver in neuroendocrine cancer progression

• Formation of positive feedback loops with oncogenic pathways like MYCN

Research shows that ELAVL3 contains three RNA recognition motifs (RRMs) with a flexible hinge region between RRM2 and RRM3, which are highly conserved across species . These structural elements contribute to its specific binding capabilities and functional properties in various cellular contexts.

What advantages do biotin-conjugated ELAVL3 antibodies offer over unconjugated versions?

Biotin-conjugated ELAVL3 antibodies provide several methodological advantages:

• Enhanced signal amplification: The biotin-streptavidin system offers one of the strongest non-covalent interactions in biology, allowing for significant signal enhancement

• Flexible detection systems: Compatible with various secondary detection methods including streptavidin-HRP, streptavidin-fluorophores, or streptavidin-gold

• Multiplexed applications: Facilitates co-staining with antibodies from the same host species

• Preserved antibody function: The small biotin molecule typically does not interfere with antibody binding capacity

• Reduced background: Can minimize non-specific interactions compared to directly labeled fluorescent antibodies

When comparing with unconjugated or other conjugated versions available in the market (including PE-conjugated ELAVL3 antibodies at $705.00) , biotin-conjugated antibodies often provide an optimal balance between sensitivity and flexibility for various experimental approaches.

What are the primary applications for ELAVL3 antibodies in neuroscience?

ELAVL3 antibodies serve various critical applications in neuroscience research:

Gene Ontology analysis reveals that ELAVL3-bound transcripts are enriched in neuron-specific processes including neuron projection development and synapse organization . This makes ELAVL3 antibodies particularly valuable for studies examining neuronal differentiation, maturation, and function.

How can biotin-conjugated ELAVL3 antibodies be utilized to investigate the ELAVL3/MYCN feedback loop in cancer?

Recent research has identified a critical positive feedback loop between ELAVL3 and MYCN in neuroendocrine prostate cancer . Biotin-conjugated ELAVL3 antibodies can be strategically employed to investigate this relationship:

• ChIP-seq analysis: Examine MYCN binding at the ELAVL3 promoter region, particularly in segments identified as responsive to MYCN (Segments A, B, and C of the ELAVL3 promoter)

• RNA immunoprecipitation: Isolate ELAVL3-bound RNA complexes to confirm direct binding to MYCN mRNA, focusing on the 3'-UTR region containing AU-rich elements

• Proximity ligation assays: Visualize and quantify protein-protein interactions between ELAVL3 and components of the transcriptional machinery

• Dual-luciferase reporter assays: Measure the impact of ELAVL3 on MYCN-dependent transcriptional activity

Research has shown that ELAVL3 binds to the 3'-UTR of MYCN mRNA, with sequences between positions 1 and 102 being the most likely binding domain . This interaction stabilizes MYCN mRNA, contributing to sustained MYCN expression and creating a self-reinforcing oncogenic circuit that could be therapeutically targeted.

What considerations should be made when designing experiments to study ELAVL3's role in neuroendocrine differentiation?

When investigating ELAVL3's role in neuroendocrine differentiation, particularly in prostate cancer, several experimental considerations are critical:

• Model selection: Choose appropriate cellular models, such as:

  • NEPC-derived cell lines (NCI-H660, LASCPC-01) with high basal ELAVL3 expression

  • Engineered adenocarcinoma models (LNCaP/AR/shTP53/shRB1 or MYCN-overexpressing cells)

  • Patient-derived xenografts that recapitulate neuroendocrine phenotypes

• Expression validation: Confirm ELAVL3 expression levels and correlation with neuroendocrine markers (SYP, CHGA, CHGB)

• Pathway analysis: Monitor activation of associated signaling cascades, particularly PI3K/AKT/mTOR pathway components through phosphorylation status of AKT and S6

• Functional readouts: Assess:

  • Cell proliferation and apoptosis rates

  • Therapy resistance (e.g., enzalutamide sensitivity)

  • Lineage marker expression (neuroendocrine vs. luminal)

  • Metastatic potential

• RNA-binding analyses: Employ RIP-seq to identify ELAVL3-bound transcripts that drive the neuroendocrine phenotype

Evidence shows that ELAVL3 overexpression alone can induce neuroendocrine phenotypes in prostate adenocarcinoma, while its knockdown reduces expression of neuroendocrine-related genes and resensitizes cells to therapy .

How do different structural domains of ELAVL3 contribute to its function, and how can antibodies help characterize these?

ELAVL3 contains three critical RNA recognition motifs (RRMs) and a hinge region, each contributing differently to its functionality:

Research indicates that deletion of RRM domains impacts ELAVL3's ability to bind and stabilize target mRNAs . Domain-specific biotin-conjugated antibodies can help isolate and characterize protein complexes associated with each functional domain, providing insights into the structural basis of ELAVL3's regulatory functions.

What validation steps are essential before using a biotin-conjugated ELAVL3 antibody in critical experiments?

Before employing a biotin-conjugated ELAVL3 antibody in pivotal experiments, thorough validation is crucial:

• Specificity validation:

  • Western blot analysis using positive controls (brain tissue, NCI-H660 cells) and negative controls

  • Competitive blocking with recombinant ELAVL3 protein, which has been shown to effectively eliminate specific staining

  • Comparison with other validated ELAVL3 antibodies from different clones

• Performance in target applications:

  • Titration experiments to determine optimal concentration for each application

  • Verification across multiple experimental methodologies (IHC, IF, WB, ELISA)

  • Cross-reactivity assessment with other ELAV family members (ELAVL1, ELAVL2, ELAVL4)

• Species reactivity confirmation:

  • Test with human samples (primary focus)

  • Validate cross-reactivity with mouse and rat samples if comparative studies are planned

• Biotin conjugation quality control:

  • Determination of biotin:antibody ratio using HABA assay

  • Assessment of storage stability under various conditions

  • Evaluation of potential steric hindrance affecting epitope recognition

Remember that immunohistochemistry specificity can be verified by blocking epitope with recombinant ELAVL3 protein, as demonstrated in previous studies .

What are the recommended sample preparation protocols for optimal ELAVL3 detection?

Optimal ELAVL3 detection requires meticulous sample preparation tailored to the experimental context:

For immunohistochemistry (paraffin-embedded tissues):

• Fix tissues in 10% neutral buffered formalin for 24-48 hours

• Process and embed in paraffin following standard protocols

• Section at 4-5 μm thickness

• Perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0)

• Block endogenous biotin using avidin-biotin blocking kit

• Apply biotin-conjugated ELAVL3 antibody at optimized dilution (typically 1:100-1:500)

• Visualize using streptavidin-HRP and DAB substrate

• Counterstain with hematoxylin

For Western blot:

• Extract proteins using RIPA buffer supplemented with RNase inhibitors

• Include phosphatase and protease inhibitors to preserve post-translational modifications

• Quantify protein concentration using BCA assay

• Load 20-40 μg protein per lane

• Separate on 10-12% SDS-PAGE gel

• Transfer to PVDF membrane

• Block with 5% non-fat milk or BSA

• Apply biotin-conjugated ELAVL3 antibody (typically 1:1000-1:2000 dilution)

• Detect using streptavidin-HRP and enhanced chemiluminescence

These protocols have been adapted from successful ELAVL3 detection methods demonstrated in previous studies examining ELAVL3 in neuroendocrine prostate cancer tissues .

What controls should be included when using biotin-conjugated ELAVL3 antibodies?

Rigorous experimental design requires appropriate controls when working with biotin-conjugated ELAVL3 antibodies:

Positive controls:

• Neural tissue samples (cerebral cortex)

• Cell lines with confirmed high ELAVL3 expression (NCI-H660, LASCPC-01)

• Recombinant ELAVL3 protein for Western blot

• ELAVL3-overexpressing transfected cells

Negative controls:

• Non-neural tissues (muscle, liver)

• Cell lines with minimal ELAVL3 expression

• ELAVL3 knockout/knockdown samples using validated shRNAs

• Secondary-only controls (omitting primary antibody)

Technical controls:

• Endogenous biotin blocking controls

• IgG isotype control (rabbit IgG at matching concentration)

• Epitope blocking controls using recombinant ELAVL3

• Dilution series to establish optimal antibody concentration

Specificity controls:

• Cross-reactivity assessment with other ELAV family proteins

• Dual staining with antibodies targeting different ELAVL3 epitopes

• Signal comparison between biotin-conjugated and unconjugated versions

A recommended control panel would include brain tissue as positive control, skeletal muscle as negative control, and ELAVL3-knockdown cells generated using validated shRNA constructs as demonstrated in previous studies .

What are common pitfalls when using biotin-conjugated antibodies for ELAVL3 detection and how can they be addressed?

Several challenges may arise when working with biotin-conjugated ELAVL3 antibodies:

It's important to note that proper validation using epitope blocking with recombinant ELAVL3 protein has been demonstrated as an effective way to confirm staining specificity in immunohistochemistry applications .

How can biotin-conjugated ELAVL3 antibodies be integrated into multiplexed detection systems?

Biotin-conjugated ELAVL3 antibodies offer versatile integration into multiplexed detection systems:

For multiplexed immunofluorescence:

• Apply biotin-conjugated ELAVL3 antibody simultaneously with unconjugated antibodies from different species

• Detect ELAVL3 using streptavidin conjugated to a specific fluorophore (e.g., Alexa Fluor 488)

• Detect other targets using species-specific secondary antibodies with non-overlapping fluorophores

• Include DAPI for nuclear counterstaining

• Analyze using multispectral imaging systems

For chromogenic multiplexing:

• Sequential detection using tyramide signal amplification (TSA)

• Apply biotin-conjugated ELAVL3 antibody first

• Detect with streptavidin-HRP and specific chromogen (e.g., DAB)

• Perform heat-mediated antibody stripping

• Apply subsequent antibodies with different chromogens

• Counterstain and analyze

This approach allows simultaneous detection of ELAVL3 with other markers like neuroendocrine markers (SYP, CHGA, CHGB) or transcription factors (MYCN, NCAM1, EZH2) that show positive correlation with ELAVL3 expression in neuroendocrine prostate cancer .

What emerging technologies incorporate biotin-conjugated antibodies for enhanced ELAVL3 detection?

Several cutting-edge technologies leverage biotin-conjugated antibodies for superior ELAVL3 detection:

• Proximity Ligation Assay (PLA):

  • Detects ELAVL3 interactions with binding partners like MYCN

  • Provides spatial resolution of protein-protein complexes

  • Generates amplified fluorescent signals at interaction sites

• CODEX multiplexed imaging:

  • Allows for detection of 40+ proteins on a single tissue section

  • Biotin-conjugated ELAVL3 antibodies can be incorporated into antibody panels

  • Enables spatial profiling of ELAVL3 in complex tissue microenvironments

• Single-cell proteogenomics:

  • Combines protein detection with RNA sequencing

  • Biotin-conjugated ELAVL3 antibodies for protein component

  • Correlates ELAVL3 protein levels with transcriptome profiles

• Super-resolution microscopy:

  • Techniques like STORM and PALM provide nanoscale resolution

  • Biotin-streptavidin detection systems offer excellent signal-to-noise ratio

  • Reveals subcellular localization of ELAVL3 with unprecedented detail

• Mass cytometry (CyTOF):

  • Metal-tagged streptavidin detects biotin-conjugated ELAVL3 antibodies

  • Enables high-dimensional analysis with 40+ parameters

  • Ideal for phenotyping complex cell populations in neuroscience research

These technologies provide powerful new avenues for studying ELAVL3's role in neural development, function, and pathological conditions, including its emerging significance in neuroendocrine cancer progression .

How can biotin-conjugated ELAVL3 antibodies contribute to cancer research?

Biotin-conjugated ELAVL3 antibodies offer valuable tools for cancer research, particularly in neuroendocrine prostate cancer (NEPC):

• Diagnostic applications:

  • Tissue microarray analysis to assess ELAVL3 upregulation in NEPC compared to adenocarcinoma

  • Classification of tumor subtypes based on ELAVL3 expression patterns

  • Correlation with neuroendocrine markers (SYP, CHGA, CHGB) and inverse correlation with androgen receptor-linked genes (AR, KLK3, NKX3-1)

• Therapeutic target validation:

  • Evaluation of ELAVL3 inhibition effects using drugs like pyrvinium pamoate

  • Assessment of lineage plasticity from neuroendocrine to luminal phenotype following ELAVL3 targeting

  • Quantification of therapy sensitization, as ELAVL3 knockdown has been shown to enhance enzalutamide sensitivity

• Pathway analysis:

  • Investigation of ELAVL3's role in activating PI3K/AKT/mTOR signaling

  • Visualization of ELAVL3-mediated post-transcriptional regulation

  • Examination of ELAVL3/MYCN positive feedback mechanisms

• Metastasis research:

  • Tracking ELAVL3-expressing cells in circulation

  • Analyzing extracellular vesicle-mediated transfer of ELAVL3 between cells

  • Correlation with metastatic burden in experimental models

Research has demonstrated that ELAVL3 deficiency reduces both weight and volume of LASCPC-01 xenografts and inhibits cell proliferation while inducing apoptosis, highlighting its potential as a therapeutic target .

What methodological approaches can optimize ELAVL3 detection in neurological disease research?

Optimizing ELAVL3 detection in neurological disease research requires tailored methodological approaches:

For brain tissue analysis:

• Perfusion fixation protocols:

  • Transcardial perfusion with 4% paraformaldehyde

  • Post-fixation for 24 hours at 4°C

  • Cryoprotection in 30% sucrose before freezing

• Sectioning techniques:

  • Free-floating sections (40 μm) for adult brain

  • Cryosections (14-16 μm) for developmental studies

  • Paraffin sections (5 μm) for archival materials

• Signal amplification methods:

  • Tyramide signal amplification (TSA) for low abundance detection

  • ABC (avidin-biotin complex) method for chromogenic visualization

  • Streptavidin-conjugated quantum dots for photostable fluorescence

• Special considerations:

  • Autofluorescence quenching with Sudan Black B

  • Lipofuscin reduction with TrueBlack or similar reagents

  • Extended primary antibody incubation (48-72 hours at 4°C)

This methodological framework builds upon demonstrated techniques for detecting ELAVL3 in neural tissues and can be extended to the study of neurodegenerative conditions where RNA-binding protein dysfunction may play a role.

How might biotin-conjugated ELAVL3 antibodies facilitate research into novel therapeutic approaches?

Biotin-conjugated ELAVL3 antibodies offer promising avenues for therapeutic research:

• Drug discovery platforms:

  • High-throughput screening for compounds disrupting the ELAVL3/MYCN feedback loop

  • Validation of pyrvinium pamoate and other potential ELAVL3 inhibitors

  • Development of antibody-drug conjugates targeting ELAVL3-expressing cells

• Mechanism-based therapeutic strategies:

  • Targeting ELAVL3's RNA-binding activity to destabilize oncogenic mRNAs

  • Disrupting ELAVL3's interaction with mTORC1 pathway components

  • Inhibiting extracellular vesicle-mediated transfer of ELAVL3

• Biomarker development:

  • Correlation of ELAVL3 expression with therapy response

  • Potential liquid biopsy applications detecting circulating ELAVL3

  • Stratification of patients for clinical trials based on ELAVL3 status

• Combination therapy approaches:

  • Synergistic targeting of ELAVL3 and androgen receptor pathways

  • Dual inhibition of ELAVL3 and MYCN signaling

  • Combined targeting of ELAVL3 and PI3K/AKT/mTOR pathway

The discovery that pyrvinium pamoate (an FDA-approved drug) can disrupt the interaction between ELAVL3 and MYCN mRNA provides a promising drug repurposing opportunity that could be rapidly translated to clinical applications for neuroendocrine prostate cancer patients .

What technological advances may enhance the utility of biotin-conjugated antibodies in ELAVL3 research?

Emerging technologies promise to expand the capabilities of biotin-conjugated ELAVL3 antibodies:

• Advanced imaging platforms:

  • Light-sheet microscopy for whole-organ ELAVL3 mapping

  • Expansion microscopy for nanoscale resolution of ELAVL3 localization

  • Intravital microscopy for tracking ELAVL3 dynamics in living systems

• Novel conjugation strategies:

  • Site-specific biotinylation for optimal antibody orientation

  • Bioorthogonal chemistry for in situ labeling applications

  • Cleavable biotin linkers for improved signal-to-noise ratios

• Single-cell technologies:

  • Integration with spatial transcriptomics

  • CITE-seq approaches correlating ELAVL3 protein with RNA expression

  • Single-cell western blot for heterogeneity assessment

• In vivo applications:

  • Biotin-conjugated ELAVL3 antibody fragments for improved tissue penetration

  • Photoacoustic imaging of ELAVL3 distribution in preclinical models

  • Theranostic approaches combining imaging and therapeutic functions

These technological frontiers will enable researchers to address outstanding questions about ELAVL3's spatial and temporal dynamics, its role in disease progression, and its potential as a therapeutic target in conditions ranging from neurodegeneration to neuroendocrine cancers.