elavl4 Antibody

What is ELAVL4 and why is it significant in neuroscience research?

ELAVL4 (Embryonic Lethal Abnormal Vision Like 4) is a member of the Hu/ELAV-like family of RNA-binding proteins predominantly expressed in neurons, with lower expression levels in the pancreas and testis . This protein post-transcriptionally processes pre-mRNAs, adding an additional layer to gene regulation and producing diverse mRNAs and proteins . ELAVL4's significance stems from its critical roles in synaptic plasticity during learning and memory tasks, as well as its regulation of multiple Alzheimer's disease (AD) candidate genes . ELAVL4 has been shown to bind and stabilize mRNAs of proteins implicated in AD pathology, including APP, BACE1, tau, and ADAM10, making it a promising target for neurodegenerative disease research .

How do I differentiate between ELAVL4 isoforms in experimental design?

The two most common isoforms of ELAVL4 (splicing variants 1 and 2, sv1 and sv2) in the postnatal brain arise from alternative splicing of ELAVL4 pre-mRNA in the region coding for the hinge between RRM2 and RRM3 . When designing experiments to detect specific isoforms:

• Use isoform-specific PCR primers targeting the differentially spliced regions of ELAVL4

• Select antibodies raised against unique epitopes when possible

• Consider the relative abundance of different isoforms in your tissue of interest

• For Western blotting, note that sv1 and sv2 can be distinguished by their slightly different molecular weights

• If studying both isoforms, include controls that express only one isoform to validate detection specificity

For complete isoform discrimination, combining RNA-seq with protein-level detection methods provides the most comprehensive analysis of ELAVL4 variant expression in experimental models.

What is the optimal method for validating ELAVL4 antibody specificity?

Knockout validation represents the gold standard for antibody specificity validation. According to published studies, researchers have effectively validated ELAVL4 antibodies using:

• Western blot analysis comparing wild-type to ELAVL4 knockout samples, which should show complete absence of signal in the knockout

• Immunocytochemistry on wild-type versus knockout neurons, with knockout samples showing undetectable signal in Nestin and N-Cadherin-expressing radial glia in the ventricular zone

• Immunoprecipitation followed by mass spectrometry to confirm pulled-down protein identity

• Peptide competition assays to demonstrate binding specificity

• Cross-validation using multiple antibodies targeting different epitopes

The Santa Cruz Biotechnology mouse monoclonal ELAVL4 antibody (3A2, sc-5261) has been extensively validated in knockout models across multiple studies at dilutions ranging from 1:200 to 1:5000 for Western blot .

What are the optimal conditions for ELAVL4 immunohistochemistry in different neural tissues?

Successful ELAVL4 immunohistochemistry requires careful consideration of tissue preparation and staining protocols. Based on published research, these approaches yield optimal results:

For co-localization studies, ELAVL4 has been successfully co-stained with Nestin and N-Cadherin to examine expression in radial glial cells . When performing double or triple immunolabeling, careful selection of primary antibodies from different host species is essential to avoid cross-reactivity.

How should I optimize Western blot protocols for ELAVL4 detection?

ELAVL4 protein detection by Western blot requires specific considerations for optimal results:

• Protein extraction should use RIPA or NP-40 based buffers with protease inhibitors to preserve intact ELAVL4

• Include RNase inhibitors in lysis buffers when studying RNA-binding capabilities

• Use 10-12% polyacrylamide gels for optimal separation

• Santa Cruz Biotechnology mouse monoclonal (3A2) has been validated at dilutions of 1:1000-1:5000

• For phosphorylated tau co-detection studies, use phosphatase inhibitors in extraction buffers

• Normalize ELAVL4 expression to GAPDH (Proteintech, 60004-1-Ig) as demonstrated in effective studies

• For membrane transfer, use PVDF membranes with 0.45μm pore size

• Blocking with 5% non-fat milk is typically sufficient, but 5% BSA may be preferable when using phospho-specific antibodies in co-detection experiments

Researchers should always include appropriate positive controls (known ELAVL4-expressing tissues) and negative controls (ELAVL4 knockout samples when available) to validate Western blot specificity.

What are the critical considerations for RNA immunoprecipitation (RIP) using ELAVL4 antibodies?

As ELAVL4 is an RNA-binding protein, RIP is a crucial technique for investigating its function. Based on published methodologies:

• The Santa Cruz ELAVL4 antibody (sc-5261) has been successfully used for RIP on human samples

• Maintain RNase-free conditions throughout the procedure

• Cross-linking with 1% formaldehyde prior to cell lysis helps preserve in vivo RNA-protein interactions

• Use magnetic beads conjugated with appropriate secondary antibodies for cleaner precipitation

• Include negative controls such as IgG from the same species as the primary antibody

• Validate RIP specificity using ELAVL4 knockout samples

• For RNA analysis, consider both targeted approaches (RT-qPCR) and global approaches (RNA-seq)

• When identifying novel ELAVL4 RNA targets, validate findings with orthogonal methods such as CLIP-seq

For known ELAVL4 targets such as APP, BACE1, and tau mRNAs, optimized primer sets should be designed to span exon-exon junctions to avoid genomic DNA amplification in post-RIP analysis.

How does ELAVL4 expression pattern change during brain development and in neurological disorders?

ELAVL4 exhibits dynamic expression patterns during development and disease states:

In normal development:

• ELAVL4 is enriched in the cortical plate where post-mitotic neurons reside

• It colocalizes with Nestin and N-Cadherin in radial glial end-feet in the E16 ventricular zone

• ELAVL4 mRNA levels remain unchanged between E13 and E16, suggesting translational derepression between these stages

In Alzheimer's disease contexts:

• ELAVL4 regulates multiple AD-related genes, including APP, BACE1, and tau

• Knockout of ELAVL4 significantly increases specific APP isoforms and intracellular phosphorylated tau

• Overexpression of ELAVL4 reduces the extracellular amyloid-beta (Aβ)42/40 ratio

• ELAVL4 has been identified as a "hub gene" in AD-related synaptic pathways

For accurate developmental profiling, combining immunohistochemistry with fluorescence-activated cell sorting (FACS) of specific neural cell populations provides the most comprehensive characterization of ELAVL4 expression dynamics.

What are the most common technical issues with ELAVL4 antibodies and how can they be resolved?

For neuronal tissues specifically, autofluorescence can interfere with ELAVL4 immunofluorescence detection. This can be mitigated by using Sudan Black B (0.1% in 70% ethanol) treatment for 10 minutes after secondary antibody incubation or by employing specialized autofluorescence quenching kits.

How can I reliably quantify changes in ELAVL4 expression levels across experimental conditions?

Accurate quantification of ELAVL4 requires rigorous methodological approaches:

• For Western blot quantification:

  • Normalize ELAVL4 signal to multiple housekeeping proteins (GAPDH, β-actin)

  • Use standard curves of recombinant ELAVL4 for absolute quantification

  • Implement technical triplicates from biological replicates

  • Apply statistical analysis appropriate for sample size and distribution

• For immunohistochemistry quantification:

  • Standardize image acquisition parameters (exposure, gain, offset)

  • Analyze multiple fields per section and multiple sections per sample

  • Use automated analysis software with consistent thresholding

  • Consider mean fluorescence intensity and cell counting approaches

  • Employ z-stack imaging for 3D quantification in thick tissues

• For mRNA expression analysis:

  • Design primers spanning exon-exon junctions

  • Validate primer efficiency using standard curves

  • Include multiple reference genes for normalization

  • Consider splicing variants in primer design and analysis

For published studies, samples (n=3) from three replicate experiments normalized to control conditions in the same experiment have provided statistically meaningful results .

What are the optimal approaches for studying ELAVL4's role in Alzheimer's disease pathways?

Given ELAVL4's established links to Alzheimer's disease mechanisms, specialized approaches can yield valuable insights:

• Use triplex electrochemiluminescence assays (e.g., MSD V-PLEX Aβ Peptide Panel 1 kit) to simultaneously measure levels of Aβ38, Aβ40, and Aβ42 in conditioned media from neuronal cultures with manipulated ELAVL4 expression

• Implement ELAVL4 knockout and overexpression in human induced pluripotent stem cell-derived neurons to study effects on:

  • APP isoform levels

  • Intracellular phosphorylated tau (detected with antibodies against phospho-tau Thr181)

  • Extracellular Aβ42/40 ratio

• Perform pathway and upstream regulator analyses of transcriptomic and proteomic data from neurons with altered ELAVL4 expression to identify:

  • Effects on synaptic function pathways

  • Gene expression changes downstream of APP and tau signaling

  • Regulation by insulin receptor-FOXO1 signaling

• Combine ELAVL4 manipulation with APP or tau pathology models to assess potential therapeutic relevance

The combination of cellular, molecular, and 'omics approaches provides the most comprehensive understanding of ELAVL4's role in AD pathophysiology.

How do I select the most appropriate ELAVL4 antibody for my specific research application?

Selection should be based on the specific detection method and research question:

For novel applications or unstudied tissue types, preliminary validation comparing multiple antibodies is strongly recommended. When possible, validate results with genetic approaches (siRNA knockdown or CRISPR knockout) to confirm specificity.

What controls are essential when designing experiments investigating ELAVL4's RNA-binding functions?

RNA-binding protein studies require rigorous controls:

• Experimental controls:

  • ELAVL4 knockout or knockdown samples

  • Overexpression of ELAVL4 (both sv1 and sv2 isoforms)

  • RNA-binding-deficient ELAVL4 mutants

  • IgG control for immunoprecipitation experiments

• Technical controls:

  • RNase treatment to confirm RNA-dependent interactions

  • Competitive binding assays with known RNA targets

  • Inclusion of non-target RNAs to assess specificity

  • In vitro binding assays with recombinant proteins

• Validation approaches:

  • Direct comparison of multiple methodologies (RIP-seq, CLIP-seq)

  • Orthogonal validation of binding using reporter assays

  • Structure-function analysis of binding domains

  • Cross-validation in multiple cell types/tissues

When studying specific targets like APP mRNA, include related family members as specificity controls and design experiments that can distinguish between direct and indirect effects on RNA metabolism.

How can emerging technologies enhance ELAVL4 antibody-based research?

Several cutting-edge approaches are poised to transform ELAVL4 research:

• Spatially-resolved techniques:

  • CLARITY and PACT tissue clearing methods have been successfully used with ELAVL4 antibodies to visualize complete neural architectures

  • Spatial transcriptomics combined with ELAVL4 immunostaining can map RNA-binding activity across tissue regions

• Single-cell applications:

  • Single-cell western blotting for protein expression heterogeneity

  • Combined single-cell RNA-seq and protein detection to correlate transcriptome with ELAVL4 levels

  • Microfluidic approaches for high-throughput single-cell analysis

• Live-cell imaging:

  • Antibody-derived nanobodies for live tracking of ELAVL4

  • Split-fluorescent protein complementation assays to study dynamic interactions

  • CRISPR-based tagging of endogenous ELAVL4 with fluorescent proteins

• Multi-omics integration:

  • Combined proteomic and transcriptomic analysis of ELAVL4-regulated networks

  • Integration with epigenomic datasets to understand regulatory mechanisms

  • Systems biology approaches to model ELAVL4 function in neuronal pathways

These technologies will enable more comprehensive characterization of ELAVL4's dynamic functions in health and disease.

What are the methodological considerations for studying ELAVL4 in human patient samples?

Working with human samples presents unique challenges:

• Tissue acquisition and processing:

  • Post-mortem interval significantly affects RNA quality and protein integrity

  • ELAVL4 detection is optimal in samples with PMI <12 hours

  • Flash-freezing or PAXgene fixation better preserves RNA-protein interactions than formalin

• Patient heterogeneity:

  • Account for age, sex, comorbidities, and medication history

  • Include sufficient sample sizes to accommodate population variance

  • Consider genetic background, particularly for neurodegenerative disease studies

• Technical adaptations:

  • Modified protein extraction protocols for limited sample quantities

  • Automated IHC systems for batch consistency across multiple patient samples

  • Multiplexed detection methods to maximize data from limited samples

• Validation approaches:

  • Complementary methodologies (IHC, Western blot, qPCR)

  • Comparison with matched controls

  • Correlation with clinical parameters

For studies examining ELAVL4 in Alzheimer's disease, stratifying samples by disease stage and correlating ELAVL4 levels with established biomarkers (Aβ, tau) provides the most clinically relevant insights.