ELP1 Antibody, Biotin conjugated

What is ELP1 and what biological significance does it have?

ELP1 (Elongator complex protein 1), also known as IKAP (IkappaB kinase complex-associated protein) or IKBKAP, is a critical component of the elongator complex, a six-subunit protein complex (ELP1-6) that plays essential roles in neurogenesis . The protein functions as a scaffold that may assemble active IKK-MAP3K14 complexes (IKKA, IKKB and MAP3K14/NIK), indicating its importance in signal transduction pathways . ELP1 has significant clinical relevance as mutations in the ELP1 gene are associated with familial dysautonomia (FD), a recessive neurodegenerative disease characterized by proprioceptive sensory loss . Additionally, bi-allelic alterations of ELP1 have been identified in about 14% of SHH-activated medulloblastomas, making it currently the most frequently mutated tumor-suppressor gene predisposing to this type of brain tumor .

What are the key specifications of the ELP1 Antibody, Biotin conjugated?

The ELP1 Antibody, Biotin conjugated (product code: CSB-PA011571LD01HU) is a polyclonal antibody raised in rabbits against a recombinant Human Elongator complex protein 1 fragment (amino acids 1107-1258) . This antibody is of IgG isotype and has been purified to >95% purity using Protein G chromatography . It is supplied in liquid form in a storage buffer containing 0.03% Proclin 300 as a preservative, 50% Glycerol, and 0.01M PBS at pH 7.4 . This antibody specifically targets human ELP1 (UniProt ID: O95163) and has been tested for applications in ELISA . The biotin conjugation enables detection systems that utilize streptavidin-based reagents, making it versatile for various immunological detection methods .

How should the ELP1 Antibody, Biotin conjugated be stored and handled?

For optimal preservation of antibody activity, the ELP1 Antibody, Biotin conjugated should be stored upon receipt at -20°C or -80°C . It is recommended to avoid repeated freeze-thaw cycles as these can degrade the antibody and reduce its effectiveness . For longer-term storage, it is advisable to aliquot the antibody into smaller volumes before freezing to minimize freeze-thaw cycles when using the reagent . When handling the antibody, maintain sterile conditions and use proper laboratory practices to prevent contamination. The antibody is supplied in 50% glycerol, which helps maintain stability during freeze-thaw processes, but proper aliquoting remains essential for preserving optimal activity throughout the product's shelf life .

What applications has the ELP1 Antibody, Biotin conjugated been validated for?

The ELP1 Antibody, Biotin conjugated has been specifically tested and validated for ELISA applications according to the manufacturer's specifications . While the non-conjugated form of this antibody (CSB-PA011571LA01HU) has recommended dilutions for Western blotting (1:500-1:5000), immunohistochemistry (1:500-1:1000), immunofluorescence (1:50-1:200), and immunoprecipitation (1:200-1:2000) , the biotin-conjugated version is primarily optimized for ELISA techniques. When used in ELISA systems, this antibody enables sensitive detection of ELP1 through streptavidin-coupled detection systems, which leverage the strong biotin-streptavidin interaction for enhanced signal generation .

How can ELP1 Antibody, Biotin conjugated be integrated into proximity labeling experiments?

ELP1 Antibody, Biotin conjugated can be strategically employed in advanced proximity labeling experiments similar to those described for other biotin-conjugated systems . For such applications, researchers can utilize the biotin-conjugated antibody to target ELP1 in cellular systems, followed by streptavidin-based pulldown to identify proteins interacting with or in close proximity to ELP1. This methodology can be enhanced by combining with SILAC (stable isotope labeling by amino acids in cell culture) approaches for quantitative analysis of the interactome . Researchers should establish experimental conditions that include appropriate negative controls (such as competing unconjugated antibody at 10-fold excess) to distinguish specific from non-specific interactions . Optimal concentrations should be determined empirically, but starting with 500 nM of the biotin-conjugated antibody would be reasonable based on similar experimental systems . After incubation and cell lysis, biotinylated proteins can be captured on streptavidin-coated magnetic beads, subjected to trypsin digestion, and analyzed by high-resolution mass spectrometry to map the ELP1 interactome with high specificity and sensitivity .

What are the critical considerations for using ELP1 Antibody, Biotin conjugated in immunohistochemistry for detecting ELP1-associated medulloblastomas?

When adapting the ELP1 Antibody, Biotin conjugated for immunohistochemical detection of ELP1-associated medulloblastomas, several critical factors must be considered. Based on related research with ELP1 antibodies, tissue preparation should include formalin fixation and paraffin embedding, with sections cut at approximately 3 μm thickness . Antigen retrieval methods should be optimized specifically for ELP1 detection in brain tissue. While the referenced study used a different ELP1 antibody clone (6G9), the biotin-conjugated antibody would require a modified protocol utilizing streptavidin-HRP detection systems rather than traditional secondary antibodies .

For validation, it is essential to include known positive and negative controls based on DNA-methylation profiled medulloblastomas, particularly focusing on SHH-activated subtypes where ELP1 mutations are most prevalent (14% of cases) . The interpretation requires careful correlation with molecular data, as immunohistochemistry aims to identify tumors with bi-allelic inactivation of ELP1 resulting from germline alterations combined with chromosome 9q loss . Automated staining platforms (such as Omnis) may provide more consistent results for clinical research applications . Researchers should establish staining patterns that reliably distinguish between normal ELP1 expression and reduced/absent expression indicative of ELP1-associated tumors.

How can splicing correction of ELP1 be monitored using molecular techniques in conjunction with antibody-based detection?

Monitoring ELP1 splicing correction in experimental therapeutic approaches requires a combination of molecular and antibody-based techniques. RT-PCR can be used with human-specific ELP1 primers (such as forward 5′-CCTGAGCAGCAATCATGTG-3′ and reverse 5′-TACATGGTCTTCGTGACATC-3′) to amplify both wild-type and mutant (Δ20) ELP1 isoforms . PCR products can be separated on 1.5% agarose gels and quantified using image analysis software to determine the relative proportions of wild-type versus mutant transcripts .

To complement the transcript analysis, the ELP1 Antibody, Biotin conjugated can be used in ELISA assays to quantify protein expression levels following splicing correction therapies. The antibody can detect human ELP1 protein expressed from transgenes in model systems . For more comprehensive analysis, researchers should design experiments that correlate transcript ratios with protein expression and functional outcomes, such as restoration of proprioceptive sensory function in familial dysautonomia models . This multi-modal approach provides robust evidence of therapeutic efficacy by demonstrating correction at both the RNA and protein levels, with functional validation in appropriate disease models.

What are common causes of high background signal when using ELP1 Antibody, Biotin conjugated in ELISA?

High background signal in ELISA using the biotin-conjugated ELP1 antibody can stem from several sources. Insufficient blocking is a primary cause, as biotin-streptavidin detection systems are highly sensitive . To minimize this issue, researchers should optimize blocking conditions using 1-5% BSA or specialized blocking buffers formulated for biotin-streptavidin systems. Another common issue is endogenous biotin in biological samples interfering with specific detection. Samples should be pre-treated with streptavidin to sequester endogenous biotin before adding the biotin-conjugated antibody .

Cross-reactivity with non-target proteins can also contribute to background. Although the antibody is purified to >95% using Protein G , residual non-specific antibodies may remain. Implementing more stringent washing procedures with PBS-Tween (0.05-0.1%) and increasing wash cycles between incubation steps can significantly reduce non-specific binding . Additionally, the antibody concentration should be titrated; excessive antibody concentrations (beyond 6.0 μg/mL based on comparable systems) can increase background . Finally, degraded or aggregated antibody from improper storage can cause non-specific binding. Ensuring proper storage at -20°C or -80°C and avoiding repeated freeze-thaw cycles will help maintain specificity and minimize background issues .

How can specificity of ELP1 Antibody, Biotin conjugated be validated in experimental systems?

Validating the specificity of ELP1 Antibody, Biotin conjugated requires multiple complementary approaches. First, competitive inhibition assays should be performed using excess unconjugated ELP1 antibody or recombinant ELP1 protein (amino acids 1107-1258, matching the immunogen) to confirm signal reduction . Second, researchers should test the antibody in cell systems with genetically modified ELP1 expression—either ELP1 knockout cells (negative control) or cells overexpressing ELP1 (positive control)—to verify correlation between ELP1 expression levels and antibody signal .

Western blot analysis using the non-conjugated version of the antibody (CSB-PA011571LA01HU) can be performed in parallel to confirm that the detected protein corresponds to the expected molecular weight of ELP1 (approximately 150 kDa) . For advanced validation, immunoprecipitation followed by mass spectrometry can verify that the antibody captures authentic ELP1 protein complexes . Cross-reactivity testing against related proteins in the elongator complex (ELP2-6) should be conducted to ensure the antibody specifically recognizes ELP1 rather than other complex components . Finally, researchers should compare results with at least one alternative ELP1 antibody from a different manufacturer or targeting a different epitope to confirm consistent detection patterns across different reagents .

What modifications to standard protocols are needed when using ELP1 Antibody, Biotin conjugated for detecting low abundance ELP1 in clinical samples?

Detecting low abundance ELP1 in clinical samples with the biotin-conjugated antibody requires several strategic protocol modifications. First, implement a signal amplification system using streptavidin-poly-HRP conjugates instead of standard streptavidin-HRP to enhance detection sensitivity without increasing background . Second, extend the primary antibody incubation time to 16-18 hours at 4°C rather than standard 1-2 hour incubations to maximize antibody binding to scarce target molecules .

Sample preparation should include more rigorous antigen retrieval methods for tissue sections or enrichment steps for protein lysates, such as immunoprecipitation with unconjugated ELP1 antibody before detection with the biotin-conjugated version . Consider using tyramide signal amplification (TSA) systems compatible with biotin-streptavidin detection to further enhance sensitivity by depositing additional biotin molecules at the site of antibody binding .

Reduce non-specific binding by using specialized blocking reagents that block endogenous biotin and biotin-binding proteins, particularly important in brain and liver tissues . Optimize antibody concentration through careful titration experiments; for low abundance targets, using concentrations at the higher end of the recommended range (closer to 6.0 μg/mL based on comparable systems) may be necessary . Finally, employ digital imaging analysis and quantification methods to objectively assess subtle differences in staining intensity that might not be apparent through visual inspection alone .

How can ELP1 detection be integrated into studies of familial dysautonomia pathophysiology?

ELP1 detection using the biotin-conjugated antibody can be strategically integrated into familial dysautonomia (FD) research through multiple approaches. This antibody can quantify ELP1 protein levels in patient-derived cells to correlate protein expression with disease severity and specific splice mutations . Researchers can employ the antibody in tissue microarray analysis of neuronal tissues from FD models to map the spatial distribution of ELP1 expression across different neuronal populations, particularly focusing on proprioceptive sensory neurons most affected in the disease .

For therapeutic development, the antibody can monitor ELP1 protein restoration following experimental treatments aimed at correcting splicing defects or enhancing expression of functional ELP1 . When combined with RT-PCR analysis of wild-type versus mutant (Δ20) transcript ratios, the antibody-based protein detection provides a comprehensive assessment of therapeutic efficacy at both RNA and protein levels . Additionally, the biotin conjugation enables multiplexed immunofluorescence studies where ELP1 detection can be combined with markers of neuronal development, degeneration, or function to elucidate how ELP1 deficiency disrupts specific neuronal circuits and processes . In drug discovery pipelines, high-throughput ELISA screening using this antibody can identify compounds that enhance ELP1 expression or stability, potentially revealing novel therapeutic candidates for FD treatment .

What insights can be gained from studying the ELP1 interactome in different cellular contexts?

Studying the ELP1 interactome across different cellular contexts using the biotin-conjugated antibody can reveal critical insights into ELP1's diverse biological functions. In neural progenitor cells, mapping ELP1 interactions can illuminate its role in neurogenesis and developmental processes affected in familial dysautonomia . By comparing ELP1 interactomes in normal versus SHH-activated medulloblastoma cells, researchers can identify altered protein interactions that contribute to tumor suppressor function loss and oncogenesis .

The biotin-conjugated antibody enables proximity labeling approaches when combined with streptavidin-based pulldowns and mass spectrometry, providing a comprehensive view of proteins physically associated with ELP1 . This approach can be enhanced through SILAC or other quantitative proteomics methods to measure dynamic changes in the interactome in response to cellular stress, differentiation signals, or therapeutic interventions . The biotin-conjugated antibody is particularly valuable for studying context-specific interactions, as it can be used in live-cell labeling followed by stringent washing steps to preserve physiologically relevant interactions while minimizing post-lysis artifacts .

This interactome analysis can reveal how ELP1 functions in both its canonical role within the elongator complex and potential non-canonical roles in IKK-related signaling pathways . By identifying key interaction partners that vary between normal and disease states, researchers can discover novel therapeutic targets that may compensate for ELP1 dysfunction or restore critical downstream pathways disrupted in ELP1-associated disorders .

What is the relationship between ELP1 expression and medulloblastoma prognosis or treatment response?

The biotin conjugation provides enhanced sensitivity for detecting subtly reduced ELP1 expression that might indicate partial loss of function, allowing for more nuanced classification beyond simple presence/absence categories . Researchers can employ the antibody in tissue microarray studies of pre- and post-treatment samples to assess whether ELP1 expression patterns change in response to standard therapies, potentially identifying it as a biomarker of treatment resistance or sensitivity .

For precision medicine approaches, the antibody can be used to screen patient-derived xenograft models or organoids to identify ELP1-deficient tumors that might respond to specific targeted therapies addressing downstream consequences of ELP1 loss . In combination with genomic analysis confirming bi-allelic ELP1 inactivation through germline mutation plus chromosome 9q loss, the antibody-based detection provides a rapid screening method to identify patients who might benefit from ELP1-targeted therapeutic strategies . This integrated approach links molecular classification with protein expression patterns, enhancing the clinical relevance of ELP1 as both a prognostic biomarker and potential therapeutic target in medulloblastoma .

How does ELP1 Antibody, Biotin conjugated compare with other detection methods for studying ELP1-associated disorders?

The ELP1 Antibody, Biotin conjugated offers distinct advantages and limitations compared to alternative detection methods for studying ELP1-associated disorders. Compared to PCR-based methods that detect ELP1 splice variants at the RNA level , this antibody provides direct information about protein expression, which may not always correlate with transcript levels due to post-transcriptional regulation. The biotin conjugation enables more sensitive detection than standard unconjugated antibodies when used with streptavidin-based amplification systems, potentially detecting lower levels of ELP1 protein in patient samples .

Mass spectrometry-based proteomics offers broader protein detection capability but typically lacks the sensitivity of antibody-based methods for specific target proteins like ELP1 . The biotin-conjugated antibody bridges these approaches when used in immunoprecipitation followed by mass spectrometry analysis of interacting partners . For optimal results in comprehensive studies of ELP1-associated disorders, researchers should implement an integrated approach combining this antibody with genomic and transcriptomic methods to correlate genetic alterations, transcript processing, and ultimate protein expression patterns .

What are optimal dilution parameters for different applications using ELP1 Antibody, Biotin conjugated?

Optimizing dilution parameters for the ELP1 Antibody, Biotin conjugated requires systematic titration across different applications. For ELISA, which is the validated application for this antibody, starting with a range of 1.0-6.0 μg/mL is recommended based on comparable biotin-conjugated antibody systems . The optimal concentration will depend on the abundance of ELP1 in the sample type, with higher concentrations needed for low-abundance samples while maintaining acceptable signal-to-background ratios .

If adapting this antibody for immunohistochemistry, despite this not being a validated application by the manufacturer, researchers should begin with a dilution range of 1:100-1:500 based on typical parameters for biotin-conjugated primary antibodies . For each new tissue type or fixation method, a dilution series should be tested to identify the concentration that provides specific staining with minimal background .

For exploratory applications in proximity labeling or protein interaction studies, concentration optimization should consider both antibody binding kinetics and potential off-target effects . At too low concentrations (<100 nM), detection sensitivity may be compromised, while excessive concentrations (>1 μM) may increase non-specific binding . A suggested starting range would be 300-700 nM based on comparable proximity labeling approaches, with competitive inhibition controls included to verify specificity .

Regardless of application, dilution optimization should include appropriate positive and negative controls, and consider sample-specific factors such as fixation method, protein abundance, and presence of potentially cross-reactive proteins . The optimal dilution should be determined empirically for each experimental system to balance detection sensitivity with specificity.