echdc1 Antibody

What is ECHDC1 and what is its primary function in cellular metabolism?

ECHDC1 (Ethylmalonyl-CoA decarboxylase) is an enzyme that plays a critical role in cellular metabolism by decarboxylating ethylmalonyl-CoA, a potentially toxic metabolite, to form butyryl-CoA. This function suggests ECHDC1 is involved in metabolite proofreading processes. Additionally, ECHDC1 exhibits methylmalonyl-CoA decarboxylase activity, albeit at lower levels compared to its primary function. The enzyme is also known as Enoyl-CoA hydratase domain-containing protein 1 or Methylmalonyl-CoA decarboxylase (MMCD) and is identified by UniProt Number Q9NTX5 .

What are the optimal storage conditions for ECHDC1 antibodies to maintain their efficacy?

ECHDC1 antibodies should be shipped at 4°C, but upon delivery, they should be aliquoted and stored either at -20°C for short-term storage or -80°C for long-term preservation. Repeated freeze-thaw cycles should be strictly avoided as they may lead to antibody degradation and loss of activity. For research applications requiring consistent antibody performance across multiple experiments, creating single-use aliquots is strongly recommended to preserve antibody integrity and binding efficiency .

How can researchers validate the specificity of ECHDC1 antibodies in their experimental systems?

Researchers should implement a multi-step validation process to confirm ECHDC1 antibody specificity:

• Western blot analysis comparing wild-type cells with ECHDC1 knockdown or knockout cells

• Immunohistochemistry with appropriate positive and negative controls

• ELISA validation using recombinant ECHDC1 protein

• Cross-reactivity testing against closely related proteins

• Competitive binding assays with the immunogen

For the most rigorous validation, implementing ECHDC1 gene silencing through shRNA approaches (as described in literature using constructs like V2LHS_175832, V2LHS_277143, or V3LHS_355397) provides definitive evidence of antibody specificity when combined with immunodetection methods .

How can ECHDC1 antibodies be utilized to investigate the relationship between ECHDC1 and ACADS deficiencies in metabolic disorders?

ECHDC1 antibodies can be employed in co-immunoprecipitation and immunoblotting experiments to examine the potential molecular interactions between ECHDC1 and ACADS (Acyl-CoA dehydrogenase short chain) proteins in both normal and pathological states. Current research indicates that ACADS and ECHDC1 deficiencies act synergistically on cellular ethylmalonic acid (EMA) excretion, suggesting a functional relationship in metabolic pathways .

For comprehensive investigation, researchers should design experiments that:

• Compare cells with varying ACADS genotypes (homozygous normal, heterozygous variant, homozygous variant)

• Implement controlled ECHDC1 knockdown using validated shRNA constructs

• Quantify both protein expression levels via immunoblotting with ECHDC1 antibodies

• Correlate protein expressions with EMA levels measured by LC-MS/MS

• Assess metabolic flux through isotope tracing experiments in combination with immunoprecipitation

This multifaceted approach enables researchers to elucidate the biochemical mechanisms underlying the observed synergistic effects on EMA metabolism .

What methodologies can be used to study ECHDC1 variant proteins using antibody-based techniques?

When investigating ECHDC1 variants such as p.Met130Thr, researchers can employ the following antibody-based methodologies:

• Immunoprecipitation followed by mass spectrometry - To identify post-translational modifications or conformational changes in variant proteins

• Pulse-chase experiments with immunodetection - To assess protein stability differences between wild-type and variant ECHDC1

• Proximity ligation assays - To investigate altered protein-protein interactions

• Immunofluorescence microscopy - To determine subcellular localization differences

These approaches should be combined with functional assays measuring enzymatic activity, such as the [14C]ethylmalonyl-CoA decarboxylase assay described in the literature. For variants with reduced expression, as observed with the intronic variants c.498-36_498-33del and c.221-4_222delinsTA that show approximately 50% ECHDC1 mRNA expression compared to controls, antibody-based quantification becomes particularly important for correlation with functional outcomes .

How can researchers optimize immunodetection of ECHDC1 in fibroblast models with low endogenous expression?

Detecting ECHDC1 in fibroblast models presents challenges due to low endogenous expression levels. Researchers can implement the following optimization strategies:

• Signal amplification techniques:

  • Tyramide signal amplification (TSA)

  • Polymer-based detection systems

  • Quantum dot-conjugated secondary antibodies

• Sample enrichment methods:

  • Subcellular fractionation to concentrate mitochondrial proteins

  • Immunoprecipitation prior to immunoblotting

  • Protein concentration methods specific for low-abundance proteins

• Detection system modifications:

  • Extended primary antibody incubation (overnight at 4°C)

  • Optimized blocking solutions (5% BSA rather than milk proteins)

  • Increased antibody concentrations with validated specificity

• Complementary approaches:

  • Combine antibody detection with targeted mass spectrometry

  • Implement parallel mRNA quantification via RT-qPCR

These optimizations can help overcome the detection limitations noted in research where "it was not possible to quantify fibroblast ECHDC1 protein level by Western blot analysis or its activity by [14C]ethylmalonyl-CoA decarboxylase assay" due to low expression levels .

What is the optimal protocol for using ECHDC1 antibodies in ELISA applications?

For optimal ELISA performance with ECHDC1 antibodies, the following protocol is recommended:

• Coating phase:

  • Use recombinant ECHDC1 protein (1-301AA) at 1-10 μg/mL in carbonate buffer (pH 9.6)

  • Incubate plates overnight at 4°C

• Blocking and antibody incubation:

  • Block with 0.01M PBS containing 5% BSA for 2 hours at room temperature

  • Apply ECHDC1 polyclonal antibody at 1:500-1:2000 dilution in antibody diluent (0.01M PBS, pH 7.4 with 0.5% BSA)

  • Incubate for 2 hours at room temperature or overnight at 4°C

• Detection and development:

  • Use appropriate HRP-conjugated secondary antibody

  • Develop with TMB substrate and measure absorbance at 450nm

• Quality control measures:

  • Include standard curves using recombinant ECHDC1 protein

  • Implement appropriate negative controls (secondary antibody only, irrelevant primary antibody)

  • Analyze data using four-parameter logistic regression

This protocol has been optimized based on the antibody specifications provided by Epigentek and is compatible with the protein G purified format of the ECHDC1 polyclonal antibody .

What are the key considerations when designing ECHDC1 knockdown experiments with antibody validation?

When designing ECHDC1 knockdown experiments that incorporate antibody validation, researchers should consider:

To ensure reproducibility across different cell models, validation should be performed in multiple cell lines as described in research where "Knockdown efficiency in the three fibroblast cell lines was initially tested using RT-qPCR with all five shRNAs, targeting ECHDC1" .

How should researchers approach troubleshooting inconsistent results when using ECHDC1 antibodies in different cell types?

When encountering inconsistent results across different cell types with ECHDC1 antibodies, a systematic troubleshooting approach should be implemented:

• Cell-specific expression analysis:

  • Quantify baseline ECHDC1 expression in each cell type via RT-qPCR

  • Use multiple reference genes (e.g., GAPDH, POP4) for accurate normalization

  • Consider isoform-specific detection (all five ECHDC1 isoforms may be differentially expressed)

• Protocol optimization by cell type:

  • Adjust lysis conditions based on subcellular localization patterns

  • Modify blocking reagents to address cell-specific background issues

  • Adjust antibody concentrations based on expression levels

• Sample preparation considerations:

  • For cells with high lipid content, incorporate additional clarification steps

  • Adjust protein extraction protocols based on cell architecture

  • Consider native vs. denatured protein detection requirements

• Validation across detection methods:

  • Compare results between Western blot, immunofluorescence, and flow cytometry

  • Implement spike-in controls with recombinant protein

  • Consider alternative antibody clones or epitope targets

This systematic approach addresses the heterogeneity in ECHDC1 expression observed across different cell models and genetic backgrounds, particularly important when studying cells with different ACADS genotypes (625G/G, 625G/A, 625A/A) .

How can ECHDC1 antibodies be used to investigate the relationship between ECHDC1 function and ethylmalonic aciduria?

ECHDC1 antibodies can be instrumental in elucidating the molecular basis of ethylmalonic aciduria through several specialized applications:

• Immunohistochemical profiling:

  • Compare ECHDC1 expression patterns in tissue samples from patients with ethylmalonic aciduria versus healthy controls

  • Correlate expression patterns with urinary EMA levels (>20 mmol/mol creatinine being clinically significant)

• Structure-function analysis:

  • Immunoprecipitate wild-type and variant ECHDC1 (e.g., p.Met130Thr) for structural and functional comparisons

  • Assess protein-protein interactions that may be disrupted in disease states

• Metabolic pathway mapping:

  • Use proximity labeling techniques with ECHDC1 antibodies to identify novel interaction partners

  • Combine with metabolomic profiling to create comprehensive pathway maps

• Therapeutic development support:

  • Evaluate potential therapeutic approaches by monitoring ECHDC1 expression and localization

  • Screen for compounds that stabilize ECHDC1 variants using antibody-based detection methods

These applications are particularly relevant for investigating cases where unexplained high levels of EMA are present but causal genetic variants have not been identified in the majority of patients (as seen in research where only 3 out of 82 individuals were found to have heterozygous variants in ECHDC1) .

What controls should be included when validating ECHDC1 antibodies for research on splicing variants?

When validating ECHDC1 antibodies for research involving splicing variants such as c.221-4_222delinsTA or c.498-36_498-33del, researchers should implement a comprehensive control strategy:

This control strategy addresses the challenges in studying intronic variants like those "located immediately downstream of a branch point motif" that may affect expression levels without creating misspliced transcripts that could be detected by sequence analysis .

How might multiplex immunoassays incorporating ECHDC1 antibodies advance metabolic disorder diagnostics?

Multiplex immunoassays incorporating ECHDC1 antibodies could transform metabolic disorder diagnostics through simultaneous detection of multiple biomarkers:

• Integrated protein panel development:

  • Combine ECHDC1 antibodies with antibodies targeting related enzymes (ACADS, ETHE1, SCAD)

  • Develop ratio-based diagnostic algorithms that increase specificity for different metabolic disorders

  • Implement machine learning approaches to identify protein expression patterns associated with specific genetic variants

• Methodological advantages:

  • Reduced sample volume requirements (critical for pediatric patients)

  • Increased throughput compared to traditional metabolite analysis

  • Potential for earlier detection before metabolite accumulation reaches clinically significant levels

• Clinical implementation considerations:

  • Correlation studies between protein levels and established metabolite biomarkers (e.g., EMA levels >20 mmol/mol creatinine)

  • Longitudinal studies to establish protein expression variability in different physiological states

  • Integration with genetic testing results for comprehensive diagnostic panels

This approach could provide complementary information to current diagnostic methods like LC-MS/MS quantification of organic acids, potentially identifying at-risk individuals before metabolite abnormalities are detected .

What are the considerations for developing antibodies against specific ECHDC1 variants for research applications?

Developing variant-specific antibodies for ECHDC1 research requires careful consideration of several factors:

• Epitope selection strategy:

  • For missense variants like p.Met130Thr, generate antibodies recognizing the variant-specific amino acid sequence

  • Design peptide immunogens that maximize exposure of the variant residue

  • Consider structural context of variants based on protein modeling

• Validation requirements:

  • Demonstrate selective binding to variant vs. wild-type protein

  • Verify absent or minimal cross-reactivity with wild-type ECHDC1

  • Confirm specificity across multiple detection platforms (Western blot, IHC, IP)

• Production challenges:

  • Some variants may have subtle conformational differences requiring specialized antibody development approaches

  • Variants with reduced expression (like those with the intronic variants showing ~50% expression) may require antibodies with higher affinity

  • Consider developing antibodies against specific ECHDC1 isoforms (from the five reported isoforms)

• Application optimization:

  • Develop specific protocols for each variant-specific antibody

  • Establish appropriate positive controls (recombinant variant proteins)

  • Define optimal buffer conditions that maximize specificity

Variant-specific antibodies would be particularly valuable for investigating the three heterozygous variants identified in research (c.389T>C/p.Met130Thr, c.221-4_222delinsTA, and c.498-36_498-33del) associated with elevated urinary EMA levels .

How can researchers integrate ECHDC1 antibody-based methods with metabolomic approaches for comprehensive metabolic pathway analysis?

The integration of ECHDC1 antibody-based detection methods with metabolomic analyses offers a powerful approach to comprehensively investigate metabolic pathways, particularly in ethylmalonic acid metabolism disorders:

• Correlation analysis protocol:

  • Quantify ECHDC1 protein expression using validated antibodies

  • Simultaneously measure relevant metabolites (ethylmalonic acid, methylmalonic acid, butyryl-CoA) by LC-MS/MS

  • Analyze data using multivariate statistical methods to identify protein-metabolite relationships

• Perturbation studies:

  • Implement ECHDC1 knockdown with shRNA constructs (achieving 50-60% reduction)

  • Monitor changes in both protein levels and metabolite profiles

  • Challenge systems with pathway inducers (e.g., sodium butyrate at 5mM for 24 hours)

• Multi-omics data integration:

  • Combine proteomics, metabolomics, and transcriptomics data

  • Create computational models of ECHDC1-dependent pathways

  • Validate model predictions with targeted antibody-based experiments