Ecel1 Antibody

What is ECEL1 and why is it significant in neurological research?

ECEL1 (Endothelin Converting Enzyme-Like 1) is a membrane-bound metalloprotease and one of the seven members of the M13 family. It is predominantly expressed in the central nervous system and plays a critical role in the nervous regulation of the respiratory system . ECEL1 contributes to the degradation of peptide hormones and is involved in the inactivation of neuronal peptides .

The significance of ECEL1 in research has increased substantially since it was identified as a gene responsible for distal arthrogryposis (DA), a congenital contracture disorder . ECEL1-mutated DA is typically characterized by ocular phenotypes in addition to congenital limb contractures. Studies using knock-in mouse models have demonstrated that ECEL1/DINE mutations lead to motor innervation defects as primary causes in these disorders .

What are the key differences between ECEL1 and DINE terminologies?

ECEL1 and DINE refer to the same protein in different species. While ECEL1 (Endothelin Converting Enzyme-Like 1) is the human designation, DINE (Damage-Induced Neuronal Endopeptidase) is the term used for the rodent ortholog . This distinction is important when selecting antibodies and interpreting cross-species studies.

Species sequence homology analysis reveals approximately 94% homology between mouse and rat DINE, and about 88% homology between rat DINE and human ECEL1 . When selecting antibodies, it's crucial to verify species reactivity, as some antibodies may recognize ECEL1/DINE across multiple species due to this high sequence conservation, while others might be species-specific.

What molecular characteristics should researchers expect when detecting ECEL1?

ECEL1 has a calculated molecular weight of 88 kDa , but researchers should anticipate detecting a band of approximately 95 kDa in Western blot applications due to heavy glycosylation . The protein has two isoforms produced by alternative splicing and three glycosylation sites, which can affect detection and apparent molecular weight.

For experimental validation, consider the following approach:

• Use 5-20% gradient SDS-PAGE for optimal protein separation

• Load approximately 50 μg of protein per lane

• Transfer to PVDF membranes for optimal protein capture

• Include glycosidase treatment controls to confirm glycosylation status

• Use GAPDH or similar housekeeping proteins as loading controls

How should researchers select the appropriate ECEL1 antibody for their specific application?

When selecting an ECEL1 antibody, researchers should consider several key factors:

For example, the antibody 14222-1-AP targets ECEL1 in ELISA applications and shows reactivity with human, mouse, and rat samples , while the ABIN6735114 antibody specifically recognizes the N-terminal region of rat DINE with cross-reactivity to mouse and human due to sequence homology .

What are the optimal protocols for detecting ECEL1 using Western blotting?

For successful Western blot detection of ECEL1, researchers should implement the following optimized protocol:

• Sample preparation:

  • Prepare protein extracts in appropriate lysis buffers containing protease inhibitors

  • For membrane proteins like ECEL1, consider specialized extraction methods

• Protein separation and transfer:

  • Use 5-20% gradient SDS-PAGE for optimal separation

  • Load 50 μg of protein sample per lane

  • Transfer to PVDF membranes using standard transfer protocols

• Blocking and antibody incubation:

  • Block membranes with 2% ECL blocking reagent

  • Incubate with primary antibody (e.g., goat anti-DINE at 1:500 dilution) at 4°C overnight

  • Wash thoroughly and incubate with appropriate HRP-conjugated secondary antibody

• Signal detection and analysis:

  • Develop using enhanced chemiluminescence (ECL) detection systems

  • Normalize to housekeeping proteins like GAPDH (1:5000 dilution)

  • Repeat experiments at least three times to ensure reproducibility

For glycosylation studies, include parallel samples treated with appropriate glycosidases to demonstrate shifts in molecular weight that confirm glycosylation status.

How can researchers effectively study ECEL1 mutations associated with distal arthrogryposis?

Studying ECEL1 mutations linked to distal arthrogryposis requires a multifaceted approach:

• Generation of knock-in mouse models:

  • Design CRISPR/Cas9 tools targeting specific mutations (e.g., G607S, C760R)

  • Create target-specific sgRNA and appropriate DNA templates

  • Verify genotypes through sequencing and perform off-target analyses

  • Cross with reporter lines like Hb9::eGFP for motor neuron visualization

• Phenotypic characterization:

  • Compare morphological phenotypes between different mutant lines

  • Assess axonal arborization defects in limb muscles

  • Evaluate guidance defects of specific nerves (e.g., abducens nerves)

  • Document developmental timing of phenotype emergence

• Molecular consequence analysis:

  • Evaluate DINE mRNA expression levels in motor neurons

  • Assess protein localization differences between mutations

  • For example, G607S mutations may show decreased or absent expression, while C760R mutations may show normal expression but abnormal localization

• Statistical analysis:

  • Apply appropriate statistical tests based on data distribution (e.g., t-tests for normally distributed data, Mann-Whitney U test for non-normal distributions)

  • For comparisons of three or more groups, use ANOVA or Kruskal-Wallis followed by post-hoc tests

  • Consider p < 0.05 as statistically significant

What immunohistochemistry approaches yield optimal results for ECEL1/DINE localization?

For optimal visualization of ECEL1/DINE in neuronal tissues, implement the following immunohistochemistry protocol:

• Tissue preparation:

  • Fix embryonic tissues (e.g., E12.5 mouse embryos) in 4% paraformaldehyde at 4°C for 2 hours

  • Immerse in PBS containing 30% sucrose for two days for cryoprotection

  • Embed in optimal cutting temperature (OCT) compound

  • Prepare 20 μm cryostat sections

• Immunostaining procedure:

  • Rinse sections three times in PBS

  • Permeabilize by immersion in absolute methanol for 6 minutes at -30°C

  • Rinse in PBS for 30 minutes

  • Block in 0.3% Triton X-100 and 0.2% bovine serum albumin in PBS

  • Incubate with primary antibody (e.g., goat anti-DINE at 1:500 dilution) overnight at room temperature

  • Apply appropriate secondary antibody (e.g., Alexa Fluor 546-conjugated anti-goat at 1:500 dilution) for 50 minutes

• Imaging and analysis:

  • Visualize using confocal microscopy for high-resolution imaging

  • Perform z-stack imaging for three-dimensional analysis

  • Compare protein distribution patterns between wild-type and mutant samples

  • Assess both neuronal cell bodies and axonal projections

How do functional consequences differ between ECEL1/DINE mutations?

Recent research has revealed distinct functional consequences of different ECEL1/DINE mutations:

• Expression differences:

  • G607S mutation: DINE mRNA and protein expression is decreased or almost absent in motor neurons

  • C760R mutation: Normal DINE expression in motor neuron somata but absence in axons

• Phenotypic similarities:

  • Both mutations lead to axonal arborization defects in target muscles

  • Normal trajectory patterns from spinal cord to target hindlimb muscles are maintained

  • Both show axon guidance defects of the abducens nerves

• Mechanistic interpretation:

  • G607S mutation appears to affect transcription or mRNA stability

  • C760R mutation likely disrupts axonal transport mechanisms

  • Both lead to similar phenotypic outcomes despite different molecular mechanisms

This understanding highlights the importance of examining both expression levels and subcellular localization when characterizing ECEL1/DINE mutations in research models.

What validation methods ensure ECEL1 antibody specificity?

Ensuring antibody specificity is critical for reliable ECEL1 research. Implement these validation approaches:

• Sequence homology assessment:

  • Verify that the antibody recognizes specific species (human, mouse, rat)

  • Confirm absence of significant sequence homology with related proteins like NEP, NEPLs, or ECEs

• Application-specific validation:

  • For Western blot: Test at recommended concentrations (1-10 μg/mL with ECL detection)

  • For ELISA: Validate at appropriate dilutions (1:10000-1:100000) using 50-100 ng control peptide/well

• Controls and competition assays:

  • Include positive controls (tissues known to express ECEL1)

  • Employ negative controls (tissues not expressing ECEL1)

  • Perform peptide competition assays using the immunogenic peptide

  • Compare results using antibodies targeting different epitopes

• Knockout/knockdown validation:

  • When available, use ECEL1/DINE knockout or knockdown models as negative controls

  • Compare signal patterns between wild-type and mutant tissues

How does ECEL1/DINE contribute to motor neuron development and function?

ECEL1/DINE plays a crucial role in motor neuron development, particularly in axonal arborization and neuromuscular junction formation:

• Axon guidance and arborization:

  • ECEL1/DINE is essential for proper axonal arborization of motor nerves in target muscles

  • Mutations lead to defects in terminal branching while maintaining normal trajectory patterns from spinal cord to target muscles

• Neuromuscular junction formation:

  • Proper motor innervation requires ECEL1/DINE function

  • Defective innervation in ECEL1/DINE mutants leads to congenital contractures

• Respiratory function:

  • ECEL1/DINE has been proposed to play a role in the nervous regulation of the respiratory system

  • This function correlates with respiratory phenotypes observed in some patients with ECEL1 mutations

• Developmental timing:

  • ECEL1/DINE function is particularly critical during embryonic development

  • Mouse models at embryonic day 12.5 (E12.5) show clear phenotypes in motor neuron development

What technical approaches can distinguish between wild-type and mutant ECEL1/DINE in knock-in models?

Differentiating between wild-type and mutant ECEL1/DINE proteins requires specialized approaches:

• Genotyping strategies:

  • Design PCR-based genotyping assays targeting the mutation site

  • Confirm mutations through direct sequencing of the target region

  • Check for potential off-target effects using mismatch cleavage enzymes

• Expression analysis:

  • Use immunohistochemistry to assess protein expression patterns

  • Compare DINE mRNA levels through in situ hybridization or qRT-PCR

  • Evaluate protein levels through Western blotting with appropriate controls

• Subcellular localization studies:

  • Perform high-resolution imaging to compare protein distribution

  • Assess the presence of protein in neuronal cell bodies versus axons

  • Use double-labeling with compartment-specific markers

• Functional assessments:

  • Document axonal arborization patterns in target muscles

  • Evaluate neuromuscular junction formation and maturation

  • Correlate molecular findings with morphological and functional phenotypes

How does glycosylation affect ECEL1 detection and analysis?

Glycosylation significantly impacts ECEL1 detection and requires specific technical considerations:

• Molecular weight variations:

  • The calculated molecular weight of ECEL1 is 88 kDa

  • Due to heavy glycosylation, ECEL1 is typically detected at approximately 95 kDa in CHO cells by Western blot

  • Glycosylation status may vary between tissues and experimental conditions

• Glycosidase treatment approaches:

  • For consistent analysis, treat protein samples with appropriate glycosidases

  • Separate treated and untreated samples using 5-20% gradient SDS-PAGE

  • Compare band shifts to assess glycosylation status

• Experimental design implications:

  • Include positive controls with known glycosylation patterns

  • Consider developmental and tissue-specific differences in glycosylation

  • Evaluate how mutations might affect glycosylation site accessibility or utilization

• Interpretation considerations:

  • Multiple bands may represent differentially glycosylated forms rather than degradation products

  • Changes in apparent molecular weight may indicate altered post-translational processing

  • Correlate glycosylation status with functional parameters when possible

What statistical approaches are appropriate for analyzing ECEL1/DINE mutation effects?

• Data distribution assessment:

  • Test data for normal distribution and equal variance

  • For normally distributed data, use parametric tests (t-tests, ANOVA)

  • For non-normally distributed data, employ non-parametric alternatives

• Two-sample comparisons:

  • For normally distributed data with equal variance: two-tailed Student's t-test

  • For normally distributed data with unequal variance: Welch's t-test

  • For non-normally distributed data: Mann-Whitney U test

• Multiple sample comparisons:

  • For normal distributions: one-way ANOVA followed by appropriate post-hoc tests

  • For non-normal distributions: Kruskal-Wallis test followed by the Steel-Dwass test

  • Consider p < 0.05 as statistically significant

• Experimental repetition:

  • Repeat experiments at least three times to ensure reproducibility

  • Present data as mean ± standard deviation or standard error

  • Use appropriate software for statistical analysis (e.g., Statcel 3)

How can researchers design comprehensive phenotypic comparisons between different ECEL1 mutations?

For thorough phenotypic comparison between different ECEL1/DINE mutations, implement this experimental design:

• Genetic model development:

  • Generate multiple knock-in mouse lines with different mutations (e.g., G607S, C760R)

  • Use consistent genetic backgrounds to minimize confounding variables

  • Include wild-type controls from the same background

  • Consider crossing with reporter lines for visualization (e.g., Hb9::eGFP)

• Multi-level phenotypic assessment:

  • Molecular level: mRNA expression, protein levels, subcellular localization

  • Cellular level: neuronal morphology, axonal guidance, arborization patterns

  • Tissue level: muscle innervation, neuromuscular junction formation

  • Organism level: limb contractures, respiratory function, ocular phenotypes

• Developmental timeline analysis:

  • Examine phenotypes at multiple developmental timepoints

  • Document the onset and progression of abnormalities

  • Correlate developmental defects with functional outcomes

• Comparative analysis framework:

  • Use consistent methodologies across all models

  • Document both similarities and differences between mutation effects

  • Correlate mouse findings with human patient data when available

  • Develop mechanistic hypotheses based on observed phenotypic patterns