ELAVL3 Antibody, HRP conjugated

What is ELAVL3 and why is it important in research?

ELAVL3 is a neural-specific RNA-binding protein belonging to the RRM ELAV protein family. It binds to AU-rich element (ARE) sequences of target mRNAs, including VEGF mRNA . With a canonical length of 367 amino acids and molecular weight of approximately 39.5 kDa in humans, ELAVL3 has been reported to exist in up to 2 different isoforms . This protein is particularly important in neuroscience research due to its brain-specific expression pattern and its role in RNA processing during neuronal differentiation . Additionally, recent research has identified ELAVL3 as a potential factor in neuroendocrine prostate cancer, making it relevant for oncology research as well .

What detection methods are most effective with ELAVL3 antibodies?

ELAVL3 antibodies are versatile reagents used in multiple detection techniques. Western blot represents the most widely used application, with over 810 citations in scientific literature describing ELAVL3 antibody use in research . Other common applications include:

• ELISA (enzyme-linked immunosorbent assay)

• Immunofluorescence

• Immunohistochemistry (IHC)

• RNA immunoprecipitation (RIP)

For IHC applications, validation studies have demonstrated that ELAVL3 antibodies can effectively detect differential expression patterns between tissue types, as exemplified by studies showing upregulation specifically in neuroendocrine prostate cancer tissues compared to other prostate cancer types .

What are the advantages of HRP-conjugated ELAVL3 antibodies?

HRP-conjugated antibodies offer several methodological advantages for ELAVL3 detection:

• Enhanced sensitivity through enzymatic signal amplification

• Elimination of secondary antibody incubation steps, reducing protocol time and potential background

• Compatibility with multiple detection substrates (chemiluminescent, colorimetric, fluorescent)

• Ideal for techniques requiring high signal-to-noise ratios

• Useful in multiplexing with differently labeled antibodies when studying ELAVL3 alongside other proteins

When working with tissue samples, HRP-conjugated antibodies have proven particularly effective in distinguishing between benign prostate tissues, hormone-sensitive prostate cancer, castration-resistant prostate adenocarcinoma, and neuroendocrine prostate cancer .

How should I optimize Western blot protocols using ELAVL3 antibodies?

Optimizing Western blot protocols for ELAVL3 detection requires several considerations:

Sample preparation:

• For neural tissues or cell lines, use a lysis buffer containing 2% sodium dodecyl sulfate, 30% glycerol, 300 mM β-mercaptoethanol, and 100 mM Tris-HCl pH 6.8

• Include protease inhibitors to prevent degradation

• Sonicate samples to shear DNA and reduce sample viscosity

Antibody dilution and incubation:

• Begin with manufacturer-recommended dilutions (typically 1:1,000 for ELAVL3 antibodies)

• For HRP-conjugated versions, optimize both antibody concentration and substrate exposure time

• Use 5% non-fat dry milk or BSA in TBST for blocking and antibody dilution

Controls and validation:

• Include recombinant ELAVL3 protein as a positive control

• Use ELAVL3-depleted samples (siRNA treated) as negative controls

• Consider using NCI-H660 or LASCPC-01 cell lysates as positive controls for high ELAVL3 expression

Optimization should be conducted systematically, changing only one parameter at a time while keeping others constant to identify optimal conditions for your specific experimental system.

What considerations are important for immunohistochemistry with ELAVL3 antibodies?

When performing IHC with ELAVL3 antibodies, researchers should consider:

Antigen retrieval methods:

• Heat-induced epitope retrieval in citrate buffer (pH 6.0) is generally effective for ELAVL3 detection

• Optimization of retrieval time may be necessary (typically 15-20 minutes)

Antibody specificity validation:

• Perform blocking studies using recombinant ELAVL3 protein to confirm antibody specificity

• Include appropriate positive control tissues (neural tissues, neuroendocrine tumors)

Quantification approaches:

• Use both staining intensity and percentage of positive cells for comprehensive evaluation

• Consider digital image analysis for objective quantification

Multiple label studies:

• For HRP-conjugated antibodies in multiplex IHC, sequential tyramine signal amplification can be used

• Carefully select chromogens with distinct colors when using multiple HRP-conjugated antibodies

Neuroendocrine prostate cancer tissues show distinctly higher ELAVL3
expression compared to other prostate tissues, making proper staining quantification essential .

How can I validate ELAVL3 antibody specificity?

Validating antibody specificity is crucial for reliable research outcomes. Recommended approaches include:

• Epitope blocking experiments: Pre-incubate the antibody with recombinant ELAVL3 protein before application to samples. This should abolish specific staining as demonstrated in published validation studies .

• siRNA/shRNA knockdown: Deplete ELAVL3 using RNA interference and confirm reduced antibody signal. This approach has been successfully used to validate antibody specificity in neural stem cells .

• Western blot analysis: Verify a single band of appropriate molecular weight (approximately 39.5 kDa) .

• Cross-reactivity assessment: Test the antibody on tissues from different species if cross-reactivity is claimed by the manufacturer .

• Comparative analysis with different antibody clones: Use multiple antibodies targeting different epitopes of ELAVL3 to confirm consistent staining patterns.

How does ELAVL3 function in RNA processing and what techniques can I use to study this?

ELAVL3 plays a critical role in RNA processing, particularly in polyadenylation site selection during neuronal differentiation . To study these functions:

RNA immunoprecipitation (RIP):

• Use ELAVL3 antibodies to isolate ELAVL3-bound RNA complexes

• Perform RIP-seq to identify ELAVL3 RNA targets on a transcriptome-wide scale

• RIP-qPCR can verify specific targets, as demonstrated in studies of MYCN transcript binding

Alternative polyadenylation analysis:

• RT-PCR with primers designed to amplify different 3' UTR variants can assess changes in polyadenylation site usage

• 4-thiouridine (4sU) labeling of nascent RNA followed by biotin pull-down allows quantification of transcript variants

Functional studies:

• siRNA-mediated depletion of ELAVL3 has been shown to cause a shift toward usage of proximal polyadenylation sites in transcripts like PES1, HNRNPA0, and GNG2

• Analysis of domain-specific functions can be performed using deletion constructs targeting specific RNA recognition motifs (RRM1, RRM2, RRM3)

Research has shown that ELAVL3 knockdown shifts polyadenylation site usage from distal to proximal sites in multiple transcripts, demonstrating its role in 3' UTR lengthening during inhibitory neuron differentiation .

What is known about ELAVL3's role in cancer and how can antibodies help study this relationship?

Recent research has revealed significant connections between ELAVL3 and neuroendocrine prostate cancer:

Expression patterns:

• ELAVL3 is specifically upregulated in neuroendocrine prostate cancer compared to other prostate cancer types

• Immunohistochemical studies with ELAVL3 antibodies show distinct expression patterns across:

  • Benign prostate tissues

  • Hormone-sensitive prostate cancer

  • Castration-resistant prostate adenocarcinoma

  • Neuroendocrine prostate cancer

Functional significance:

• ELAVL3 overexpression is sufficient to induce neuroendocrine phenotype in prostate adenocarcinoma

• ELAVL3 forms a positive feedback loop with MYCN, stabilizing MYCN mRNA through binding to its 3' UTR

• ELAVL3 deficiency reduces tumor growth in xenograft models and shifts cells from neuroendocrine to luminal phenotypes

Therapeutic implications:

• ELAVL3 knockdown increases sensitivity to enzalutamide therapy, with enhanced inhibitory effects when combined

• ELAVL3 overexpression confers resistance to enzalutamide (IC50 of 74.17 μM vs. 23.55 μM in control cells)

Antibody-based approaches including IHC, Western blotting, and RIP assays have been instrumental in elucidating these relationships. Particularly, HRP-conjugated antibodies can provide the sensitivity needed for detecting subtle expression differences between tumor subtypes.

Which domains of ELAVL3 are crucial for its function and how can they be studied?

ELAVL3 contains three RNA recognition motifs (RRMs) and a hinge region that are highly conserved across species . Studies using domain-specific deletion constructs have revealed:

Research has demonstrated that deletion of either RRM1 or RRM2 prevents ELAVL3 from stimulating MYCN expression or inducing neuroendocrine phenotypes . RIP-seq analysis comparing wildtype ELAVL3 and RRM1-deleted variants showed that RRM1 is necessary for binding to MYCN transcripts .

When designing experiments to study domain-specific functions, using antibodies that recognize different regions of ELAVL3 can provide complementary information about protein interactions and conformational changes.

What are common issues with ELAVL3 antibody applications and how can they be addressed?

When working with ELAVL3 antibodies, researchers frequently encounter several challenges:

Background signal issues:

• Problem: Non-specific background staining in IHC or Western blot

• Solution: Increase blocking time/concentration, optimize antibody dilution, include additional wash steps

Inconsistent results across experiments:

• Problem: Variable signal intensity between replicates

• Solution: Standardize protein loading, optimize antibody concentration, ensure consistent incubation times and temperatures

Cross-reactivity concerns:

• Problem: Potential cross-reactivity with other ELAV family members (ELAVL1/HuR, ELAVL2/HuB, ELAVL4/HuD)

• Solution: Validate antibody specificity using recombinant proteins or knockout/knockdown systems

Epitope masking:

• Problem: Protein-protein interactions or post-translational modifications may mask the epitope

• Solution: Try different fixation methods, alternative antibody clones, or different epitope targets

Signal detection limitations:

• Problem: Weak signal from low-abundance targets

• Solution: For HRP-conjugated antibodies, use high-sensitivity substrates, increase antibody concentration, or implement signal amplification methods

How should I design controls for experiments using ELAVL3 antibodies?

Robust experimental design requires appropriate controls:

Positive controls:

• Tissues/cells known to express ELAVL3 (neural tissues, NCI-H660 or LASCPC-01 cell lines)

• Recombinant ELAVL3 protein for Western blot standardization

Negative controls:

• ELAVL3 knockout or knockdown samples

• Tissues known to lack ELAVL3 expression

• Antibody diluent without primary antibody

Specificity controls:

• Pre-adsorption with recombinant ELAVL3 protein

• Isotype-matched control antibodies

• Competitive binding experiments

Quantitative controls:

• Standard curves with recombinant protein for quantitative applications

• Housekeeping proteins (like actin) for Western blot normalization

• Calibrated reference standards for absolute quantification

For HRP-conjugated antibodies specifically, include enzyme activity controls to verify conjugate functionality and stability over time.

How might ELAVL3 antibodies contribute to therapeutic development?

ELAVL3's role in neuroendocrine prostate cancer suggests several therapeutic applications:

Biomarker development:

• ELAVL3 antibodies could help identify patients with neuroendocrine features who might benefit from targeted therapies

• Monitoring ELAVL3 expression could track therapeutic response or disease progression

Target validation:

• Antibody-based studies have established the ELAVL3/MYCN feedback loop as a potential therapeutic target

• Combining ELAVL3 downregulation with enzalutamide treatment shows enhanced therapeutic effects

Drug screening:

• HRP-conjugated ELAVL3 antibodies could facilitate high-throughput screening assays to identify compounds disrupting ELAVL3 binding to target RNAs

• Compounds targeting ELAVL3-MYCN interactions might represent novel therapeutic approaches

Research has demonstrated that ELAVL3 deficiency increases sensitivity to enzalutamide therapy, suggesting potential combination therapy approaches . Further studies using well-validated antibodies will be critical in translating these findings into clinical applications.

What emerging technologies can enhance ELAVL3 antibody applications?

Several cutting-edge technologies show promise for expanding ELAVL3 research:

Proximity ligation assays:

• Can detect protein-protein interactions involving ELAVL3 in situ

• Useful for studying components of ELAVL3-containing ribonucleoprotein complexes

Mass cytometry (CyTOF):

• Allows simultaneous detection of multiple proteins including ELAVL3

• Can reveal heterogeneity in expression across cell populations

Spatial transcriptomics:

• Combined with immunohistochemistry can correlate ELAVL3 protein expression with transcriptome-wide effects

• Provides insights into tissue-specific functions

CRISPR-based approaches:

• Gene editing of ELAVL3 binding sites can validate direct RNA targets

• Domain-specific mutagenesis can dissect structure-function relationships

Single-cell proteomics:

• Can reveal cell-to-cell variability in ELAVL3 expression and function

• Particularly relevant for heterogeneous tissues like tumors