ELP1 Antibody

What is ELP1 and why is it important in research?

ELP1, also known as IKBKAP (IκB kinase complex-associated protein), functions as a critical subunit of the Elongator complex. Initially identified as a scaffolding protein within the NFκB signaling pathway, ELP1 contains several WD40 domains essential for Elongator complex formation . Research significance stems from its role in neurological disorders, as mutations in ELP1 cause Familial Dysautonomia (FD), an autosomal recessive neurodegenerative disorder . ELP1 mutations affect transcription elongation of genes involved in cell motility and neuronal development, potentially underlying the neuropathology observed in FD patients . Additionally, bi-allelic alterations of ELP1 have been identified in medulloblastoma (MB), where it functions as a tumor-suppressor gene and is currently recognized as the most frequent gene predisposing to MB in certain subtypes .

What are the available types of ELP1 antibodies and their specifications?

Commercial ELP1 antibodies include polyclonal antibodies derived from rabbits targeting various epitopes of the protein. For example, Cell Signaling Technology offers an ELP1/IKBKAP antibody (#5071) with reactivity to human and monkey samples, demonstrating endogenous sensitivity . This particular antibody detects ELP1 protein at approximately 150 kDa and is suitable for Western Blotting (1:1000 dilution) and Immunoprecipitation (1:50 dilution) . For immunohistochemistry applications, clone 6G9 (1:50 dilution; Sigma-Aldrich) has been validated for detection of ELP1 in formalin-fixed, paraffin-embedded tissue samples . When selecting an antibody, researchers should consider the specific application, target species, and epitope recognition requirements for their experimental design.

What are the validated protocols for ELP1 immunohistochemistry in clinical samples?

For ELP1 immunohistochemistry in clinical samples, protocols have been validated particularly for medulloblastoma classification. A standardized approach includes:

• Sample preparation: Use 3 μm-thick sections of formalin-fixed, paraffin-embedded tissue samples .

• Antibody selection: Employ the ELP1 antibody clone 6G9 at 1:50 dilution (Sigma-Aldrich) .

• Staining protocol: Perform immunostaining on an automated platform (e.g., Omnis automate) .

• Controls integration: Include intra-tumoral vessels as positive internal controls, which maintain ELP1 expression even in tumors with ELP1 loss .

• Interpretation: Complete loss of cytoplasmic ELP1 staining in all tumor cells (with preserved staining in internal controls) indicates potential ELP1-associated medulloblastoma .

• Validation: Confirm findings with molecular analyses for bi-allelic ELP1 alterations when possible .

This methodology has demonstrated 99% sensitivity and 100% specificity for identifying ELP1-associated medulloblastoma, making it a valuable diagnostic tool in neuropathology .

How can I use ELP1 antibodies for protein expression characterization during developmental stages?

For characterizing ELP1 protein expression during developmental stages, a staged approach has proven effective:

• Temporal grouping: Organize embryos or samples by developmental timepoints (e.g., every 12-24 hours during embryonic development), aligning with key developmental events such as neural crest cell migration or neuronal condensation .

• Antibody validation: Conduct control experiments to verify antibody specificity by:

  • Performing positive immunostaining with the ELP1 antibody alongside markers for relevant cell types (e.g., Sox10 for neural crest cells, Tubb3 for placode-derived neurons) .

  • Running negative controls by omitting the primary ELP1 antibody while including the same secondary antibody and additional markers .

• Co-localization studies: Combine ELP1 immunostaining with lineage-specific markers to determine expression in distinct cell populations during development .

• Quantification: Measure intensity levels across developmental stages to track dynamic expression changes.

• Functional correlation: Correlate expression patterns with known developmental events to establish potential functional roles of ELP1 at specific developmental stages.

This methodology enables robust spatio-temporal characterization of ELP1 expression throughout development, particularly in neuronal contexts.

What are the recommended approaches for validating ELP1 antibody specificity?

Validating ELP1 antibody specificity requires a multi-faceted approach:

• Positive and negative control tissues: Test antibodies on tissues/cells known to express or lack ELP1. For instance, in medulloblastoma research, comparing SHH-activated tumors (potentially ELP1-negative) with WNT-activated, group 3, and group 4 medulloblastomas (ELP1-positive) provides appropriate controls .

• Antibody omission control: Perform immunostaining without the primary ELP1 antibody but with all other reagents to identify non-specific binding of secondary antibodies. This approach validated the specificity of Elp1 antibody in developmental studies, confirming absence of signal when primary antibody was omitted .

• Genetic validation: Test antibodies on tissues/cells with known ELP1 mutations or knockdowns. For example, validating antibody specificity using Elp1 morpholino knockdowns, which demonstrated reduced Elp1 protein levels via immunoblotting .

• Molecular correlation: Correlate immunohistochemistry results with genomic or proteomic data. Studies have shown strong correlation between ELP1 immunohistochemistry and genetic findings, with 11/12 MB cases showing ELP1 protein loss also demonstrating bi-allelic ELP1 alterations .

• Cross-reference multiple antibodies: When possible, validate findings with multiple antibodies targeting different ELP1 epitopes to confirm consistent results.

These validation steps ensure reliable interpretation of experimental results using ELP1 antibodies.

How can ELP1 antibodies be utilized for detecting ELP1-associated medulloblastoma?

ELP1 immunohistochemistry (IHC) has emerged as a highly specific and sensitive diagnostic tool for identifying ELP1-associated medulloblastoma:

• Diagnostic application: ELP1 IHC detects ELP1-associated MB with 99% sensitivity and 100% specificity, providing a rapid and cost-effective screening method compared to comprehensive genetic testing .

• Interpretation criteria: Complete loss of cytoplasmic ELP1 staining in tumor cells, while maintaining expression in intra-tumoral vessels (serving as internal positive controls), indicates potential ELP1-associated medulloblastoma .

• Subgroup correlation: ELP1 loss is predominantly observed in SHH-activated medulloblastoma (21% of cases in studied cohorts), particularly in pediatric patients, and is not typically seen in WNT-activated, group 3, or group 4 medulloblastomas .

• Clinical significance: Detection of ELP1-associated medulloblastoma may indicate a related cancer predisposition syndrome, warranting additional genetic counseling and family screening .

• Implementation: ELP1 antibody IHC should be incorporated into routine neuropathology panels for medulloblastoma classification, potentially identifying patients with underlying genetic predisposition .

This application demonstrates how ELP1 antibodies can bridge molecular diagnostics and conventional pathology, providing critical information for patient management .

What are the current limitations and challenges in using ELP1 antibodies for research?

Despite their utility, researchers face several challenges when working with ELP1 antibodies:

• Cryptic alterations detection: Current antibody-based methods may indicate protein loss without identifying the underlying genetic mechanism. In one study, a case showed ELP1 protein loss via IHC without detectable bi-allelic ELP1 alteration through standard sequencing, suggesting cryptic alterations not detected by conventional methods .

• Heterogeneous expression patterns: Interpreting partial or heterogeneous staining patterns remains challenging, particularly in distinguishing technical artifacts from biologically significant expression changes.

• Cross-reactivity concerns: ELP1 antibodies may show cross-reactivity with similar proteins containing WD40 domains, potentially resulting in false positive signals in certain contexts.

• Post-translational modifications: Current antibodies may not distinguish between different post-translational modifications of ELP1, which could have distinct functional implications.

• Limited epitope coverage: Available antibodies target specific epitopes, potentially missing truncated or alternatively spliced variants of ELP1 that might retain partial function.

• Sensitivity in low-expression contexts: Detection of low-level ELP1 expression in certain cell types or developmental stages may require enhanced sensitivity beyond standard immunohistochemistry protocols.

Researchers should acknowledge these limitations when designing experiments and interpreting results, potentially employing complementary molecular techniques to validate antibody-based findings.

How do you troubleshoot discrepancies between ELP1 immunohistochemistry and genetic testing results?

When faced with discrepancies between ELP1 protein expression (by IHC) and genetic testing results, consider the following systematic troubleshooting approach:

• Verify antibody specificity: Reconfirm antibody specificity using appropriate controls. Ensure the antibody recognizes the relevant epitope that would be affected by the genetic alteration in question .

• Consider cryptic alterations: As observed in one medulloblastoma case showing protein loss without detectable genetic alteration, cryptic mechanisms might be present. These could include:

  • Deep intronic pathogenic variants

  • Complex structural variants

  • Promoter genomic alterations

  • Hypermethylation

  • Post-transcriptional regulatory mechanisms

• Employ complementary techniques: Utilize proteomic analysis to quantify ELP1 protein levels. In the discordant case documented, proteomic analysis confirmed downregulated levels of ELP1 despite normal sequencing results .

• Assess chromosomal status: Examine the loss of heterozygosity (LOH) status of chromosome 9q, which frequently accompanies ELP1 alterations. Copy-neutral LOH may result in protein loss without detectable sequence variants .

• Evaluate other Elongator complex components: Consider alterations in other Elongator complex components that might affect ELP1 stability or expression without direct ELP1 mutations.

• Technical considerations: Review technical aspects of both assays, including tissue fixation, processing artifacts, antibody penetration issues, and sequencing quality/coverage limitations.

This systematic approach can help resolve apparent discrepancies and potentially reveal novel mechanisms of ELP1 dysregulation.

How does ELP1 antibody staining pattern correlate with different mutations and their functional consequences?

The correlation between ELP1 staining patterns and specific mutations provides insight into structure-function relationships and disease mechanisms:

• Complete loss patterns: Bi-allelic inactivation of ELP1, resulting from combinations of nonsense mutations, frameshift mutations, or splice site alterations, typically presents as complete loss of cytoplasmic ELP1 staining in affected cells . For example, mutations such as p.(Gln911*), p.(Arg226Ter), and c.741-1G>T result in complete protein loss detectable by IHC .

• Mutation-specific patterns: Different mutation types may produce distinct staining patterns:

  • Truncating mutations early in the protein sequence generally result in complete protein loss

  • Missense mutations (e.g., p.(Glu541Gly), p.(Pro410Leu)) may show variable staining depending on protein stability and epitope preservation

  • Splice site mutations might produce aberrant proteins with altered localization or expression levels

• Functional correlation: In medulloblastoma research, complete loss of ELP1 protein strongly correlates with bi-allelic inactivation and demonstrates a distinct biological phenotype within SHH-activated medulloblastomas .

Understanding these correlations helps researchers interpret IHC results in the context of specific genetic alterations and their functional impacts.

What is the role of ELP1 antibodies in studying neuronal development and familial dysautonomia?

ELP1 antibodies serve as crucial tools for investigating neuronal development and understanding Familial Dysautonomia pathogenesis:

• Developmental expression mapping: ELP1 antibodies enable characterization of protein expression during critical neurodevelopmental stages. Studies have employed staged embryo grouping (every 12-24 hours) to correlate ELP1 expression with key developmental events like neural crest cell migration and neuronal condensation .

• Cell lineage studies: Combining ELP1 immunostaining with lineage markers (e.g., Sox10 for neural crest cells, Tubb3 for placode-derived neurons) helps identify specific neuronal populations dependent on ELP1 function .

• Familial Dysautonomia models: In FD research, ELP1 antibodies are essential for validating disease models by confirming reduced protein levels in knockdown/knockout systems. Morpholino-based approaches targeting ELP1 have been validated via immunoblotting to confirm efficacy in reducing ELP1 protein levels .

• Mechanistic studies: ELP1 antibodies help elucidate how mutations affect the Elongator complex formation and function. Research has demonstrated that defects in Elongator function upon ELP1 mutation impact transcription elongation of genes involved in cell motility and neuronal development, potentially explaining FD neuropathology .

• Therapeutic development: For potential FD therapies, ELP1 antibodies provide crucial tools to assess treatment efficacy in restoring protein levels or function in affected tissues.

These applications highlight how ELP1 antibodies bridge genetic findings with functional consequences in neuronal development and disease contexts.

How can researchers effectively combine ELP1 antibodies with other molecular tools for comprehensive pathway analysis?

Integrative approaches combining ELP1 antibodies with complementary molecular techniques enable comprehensive pathway analysis:

• Multi-omics integration: Combine ELP1 immunostaining with genomic and proteomic data. Studies have successfully integrated ELP1 IHC results with Next-Generation Sequencing (NGS) and proteomic quantification via data-independent acquisition methods to comprehensively characterize ELP1-associated pathologies .

• Co-immunoprecipitation studies: Utilize ELP1 antibodies for co-immunoprecipitation (recommended dilution 1:50) to identify interaction partners and investigate Elongator complex formation dynamics . This approach can reveal how specific mutations disrupt protein-protein interactions within the complex.

• Chromatin studies: Combine ELP1 antibodies with chromatin immunoprecipitation (ChIP) to investigate its role in transcriptional regulation, particularly for genes involved in cell motility and neuronal development implicated in FD pathology .

• Morpholino/CRISPR knockdown validation: Validate genetic manipulation approaches by confirming reduced ELP1 protein levels with immunoblotting using ELP1 antibodies . This provides crucial functional validation of genetic tools.

• Single-cell approaches: Integrate ELP1 immunostaining with single-cell technologies to identify cell-specific expression patterns and potential heterogeneity in disease contexts.

• Live imaging: Employ fluorescently tagged antibodies or antibody fragments to track ELP1 dynamics in living systems, particularly during development or in response to perturbations.

This integrative approach provides mechanistic insights beyond what any single technique could offer, advancing understanding of ELP1 function in development and disease.

What emerging applications of ELP1 antibodies are being developed for precision medicine?

Emerging applications of ELP1 antibodies in precision medicine represent a frontier in translational research:

• Diagnostic biomarker development: ELP1 IHC is being developed as a rapid, cost-effective diagnostic biomarker for ELP1-associated medulloblastoma, with demonstrated 99% sensitivity and 100% specificity . This approach enables efficient screening for potential germline cancer predisposition syndromes, allowing earlier intervention and family screening.

• Therapeutic response monitoring: ELP1 antibodies could potentially monitor treatment efficacy in ELP1-associated disorders by assessing protein restoration or pathway normalization following experimental therapies.

• Patient stratification: As different ELP1 mutations may have distinct functional consequences, immunohistochemical patterns might help stratify patients for tailored therapeutic approaches targeting specific pathogenic mechanisms.

• Liquid biopsy development: Research may explore detection of ELP1 protein fragments or ELP1-containing extracellular vesicles in body fluids as minimally invasive biomarkers for neurodegenerative conditions or certain cancers.

• Theranostic applications: Developing ELP1 antibody-based imaging probes could enable visualization of affected tissues in vivo, potentially guiding surgical interventions or targeted therapy delivery.

These emerging applications highlight how basic research tools are being translated into clinically relevant applications with potential impact on patient care.

How can researchers contribute to improving ELP1 antibody development and standardization?

Researchers can advance ELP1 antibody quality and standardization through several key approaches:

• Epitope mapping: Comprehensively map epitopes recognized by existing antibodies and develop new antibodies targeting functionally significant domains or mutation-prone regions of ELP1.

• Validation consortia: Participate in collaborative validation efforts to systematically compare antibody performance across multiple laboratories using standardized samples and protocols.

• Application-specific optimization: Develop and publish optimized protocols for specific applications (IHC, WB, IP) across different tissue types and fixation methods, addressing variables that affect antibody performance.

• Reference materials development: Create and share characterized reference materials with known ELP1 status (wildtype, specific mutations, or knockout) to serve as standardized controls for antibody validation.

• Cross-platform validation: Systematically compare antibody-based results with orthogonal methods (mass spectrometry, RNA-seq, genetic testing) to establish concordance rates and identify potential discrepancies.

• Open data sharing: Contribute detailed antibody validation data to public repositories and antibody registration initiatives to improve transparency and reproducibility.

• Novel antibody types: Develop recombinant antibodies with defined sequences and consistent production methods to reduce batch-to-batch variability inherent in polyclonal antibodies.

These contributions would significantly advance the reliability and utility of ELP1 antibodies for both research and clinical applications.

What are the key considerations for researchers designing experiments to investigate ELP1 function using antibody-based approaches?

Researchers designing ELP1 antibody-based experiments should consider these critical factors:

By addressing these considerations, researchers can design robust experiments that maximize the utility of ELP1 antibodies while minimizing potential pitfalls and misinterpretations.