ECEL1 Antibody, FITC conjugated

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

ECEL1 (Endothelin Converting Enzyme-Like 1) is a type II membrane-bound metalloprotease belonging to the M13 family that includes neprilysin (NEP) and endothelin-converting enzyme (ECE). It is predominantly expressed in the central nervous system and plays critical roles in:

• Motor neuron development and axonal arborization

• Neuronal peptide processing

• Respiratory system nervous regulation

• Degradation of peptide hormones

ECEL1 (also known as DINE) has gained significant research interest due to its association with distal arthrogryposis type 5D (DA5D), a congenital disorder affecting limb movement. Studies using genetically manipulated mice have demonstrated that ECEL1 is essential for proper arborization of embryonic motor axons in specific muscle subsets, including respiratory muscles .

The protein has 2 isoforms produced by alternative splicing and 3 glycosylation sites, with a calculated molecular weight of 88 kDa but often detected at approximately 95 kDa in CHO cells due to heavy glycosylation .

What applications can ECEL1 antibody, FITC conjugated support in research?

ECEL1 antibody, FITC conjugated supports several key applications in neuroscience and developmental biology research:

• ELISA: Primary application for quantitative detection of ECEL1 protein levels

• Immunohistochemistry: Visualization of ECEL1 expression patterns in tissue sections

• Immunofluorescence: Co-localization studies with other neuronal markers

• Flow cytometry: Analysis of ECEL1 expression in neuronal populations

• Developmental studies: Tracking ECEL1 expression during neuromuscular junction formation

• Disease model research: Investigating ECEL1 involvement in distal arthrogryposis

The FITC conjugation eliminates the need for secondary antibody incubation steps, reducing background and simplifying experimental workflows in fluorescence-based detection methods.

What are the key technical specifications of commercially available ECEL1 antibody, FITC conjugated?

Based on multiple manufacturers' data:

How can ECEL1 antibody, FITC conjugated be used to investigate neurodevelopmental disorders?

ECEL1 mutations are directly linked to distal arthrogryposis type 5D (DA5D), characterized by contractures of distal joints, including camptodactyly, calcaneovalgus feet, knee extension contractures, and facial features like ptosis and micrognathia .

Methodological approach for investigating DA5D pathophysiology:

• Patient-derived samples analysis:

  • Use ECEL1 antibody, FITC conjugated for immunostaining of muscle biopsies from DA5D patients

  • Compare ECEL1 localization and expression patterns with control tissues

  • Correlate protein expression with specific mutations and clinical severity

• In vitro modeling:

  • Generate cell models expressing wild-type and mutant ECEL1 (such as the novel c.535A>G (p. Lys179Glu) variant)

  • Use FITC-conjugated antibodies for live-cell tracking of protein trafficking

  • Compare subcellular localization patterns between wild-type and mutant proteins

• Structural impact assessment:

  • Combine molecular dynamics simulation data of ECEL1 mutants with antibody epitope mapping

  • The search results describe a study using MD simulation to demonstrate "remarkable constructional differences" between wild-type and novel mutant ECEL1, particularly in zinc ion binding capacity

  • Antibody binding patterns can provide experimental validation of these structural changes

What considerations are important when using ECEL1 antibody, FITC conjugated for co-localization studies?

When designing co-localization experiments with ECEL1 antibody, FITC conjugated:

• Fluorophore selection for multi-color imaging:

  • FITC emits green fluorescence (peak ~525 nm)

  • Choose companion fluorophores with minimal spectral overlap:

    • Red fluorophores (e.g., Texas Red, Cy3) for dual labeling

    • Far-red fluorophores (e.g., Cy5, Alexa Fluor 647) for triple labeling

    • Blue fluorophores (e.g., DAPI) for nuclear counterstaining

• Sample preparation optimization:

  • Fixation method affects epitope availability and FITC fluorescence

  • Recommended: 4% paraformaldehyde fixation (as used in the fusion assay protocol)

  • Avoid harsh fixatives that may denature the epitope region (AA 425-775)

• Signal validation controls:

  • Include single-color controls to assess bleed-through

  • Use FITC-only antibodies (non-ECEL1 targeting) as fluorophore controls

  • Include peptide competition controls using recombinant ECEL1 (AA 425-775)

• Photobleaching considerations:

  • FITC is more susceptible to photobleaching than some other fluorophores

  • Image FITC channels first in sequential imaging protocols

  • Consider using anti-fade mounting media with DABCO or similar components

How can researchers validate the specificity of ECEL1 antibody, FITC conjugated?

Validating antibody specificity is crucial for research reliability. For ECEL1 antibody, FITC conjugated:

• Genetic validation approaches:

  • Use CRISPR/Cas9-mediated ECEL1 knockout cells as negative controls

  • Employ ECEL1-overexpressing cells as positive controls

  • Test reactivity in cells expressing only specific domains of ECEL1 to confirm epitope specificity

• Biochemical validation methods:

  • Peptide competition assays using the immunogen (ECEL1 AA 425-775)

  • Western blot analysis to confirm recognition of the expected 88-95 kDa band (note: ECEL1 appears at ~95 kDa in CHO cells due to glycosylation)

  • Immunoprecipitation followed by mass spectrometry to confirm target identity

• Cross-reactivity assessment:

  • Test against related M13 family metalloproteases (NEP, ECE)

  • Evaluate in tissues known to be negative for ECEL1 expression

  • Confirm reactivity pattern matches known ECEL1 distribution (predominantly in central nervous system)

• Application-specific controls:

  • For ELISA: Include standard curves using recombinant ECEL1 protein

  • For fluorescence microscopy: Compare to alternative ECEL1 antibodies with different epitopes

  • For flow cytometry: Use isotype control antibodies conjugated to FITC

What is the optimal protocol for using ECEL1 antibody, FITC conjugated in immunofluorescence studies?

Based on protocols mentioned in the search results and standard practices for FITC-conjugated antibodies:

Immunofluorescence Protocol for Cell Cultures:

• Sample preparation:

  • Grow cells on glass coverslips or chamber slides

  • Fix with 4% paraformaldehyde for 10-15 minutes at room temperature

  • Wash 3× with PBS

• Permeabilization and blocking:

  • Permeabilize with 0.1-0.3% Triton X-100 in PBS for 10 minutes

  • Block with 5% normal serum (from non-rabbit species) with 1% BSA in PBS for 1 hour

• Primary antibody incubation:

  • Dilute ECEL1 antibody, FITC conjugated in blocking buffer (recommended starting dilution: 1:100)

  • Incubate overnight at 4°C in a humidified chamber protected from light

  • Wash 3× with PBS

• Counterstaining:

  • Nuclear counterstain: Incubate with DAPI (1:1000) for 5 minutes

  • For co-staining with other markers: Use antibodies with compatible fluorophores

• Mounting and imaging:

  • Mount with anti-fade mounting medium

  • Image using appropriate filter sets for FITC (excitation ~495 nm, emission ~520 nm)

  • Store slides at 4°C protected from light

Evidence from immunofluorescence applications shows successful ECEL1 detection in:

• HeLa cells (cervical cancer cell line)

• HepG2 cells (liver hepatocellular carcinoma cell line)

How should researchers optimize dilution ratios for ECEL1 antibody, FITC conjugated across different applications?

Optimizing antibody dilution is critical for balancing specific signal and background. Based on available data:

General optimization strategy:

• Start with manufacturer recommendations:

  • ELISA: Typically 1:100 to 1:500 range

  • Immunofluorescence: 1:100 has been reported successful

• Perform titration experiments:

  • Test serial dilutions (e.g., 1:50, 1:100, 1:200, 1:500, 1:1000)

  • Select dilution with highest signal-to-noise ratio

  • Note: "Optimal working dilution should be determined by the investigator"

• Application-specific considerations:

  • ELISA:

    • Higher dilutions (1:500-1:1000) often suitable for plate-based assays

    • Include standard curves with each new antibody lot

  • Immunofluorescence/IHC:

    • Typically requires lower dilutions (1:50-1:200)

    • Increase antibody concentration for weakly expressed targets

    • Decrease concentration if background is problematic

  • Flow cytometry:

    • Start with 1:100 dilution

    • Optimize based on signal separation between positive and negative populations

• Sample-specific adjustments:

  • Fresh tissues may require higher dilutions than FFPE samples

  • Cell lines with ECEL1 overexpression may allow higher dilutions

  • Different fixation methods may require adjusted concentrations

What are the best practices for storage and handling of ECEL1 antibody, FITC conjugated to maintain activity?

Proper storage and handling are crucial for maintaining antibody activity, especially for fluorophore-conjugated antibodies:

• Storage conditions:

  • Store at -20°C to -80°C as recommended by multiple manufacturers

  • Keep in the dark to prevent photobleaching of FITC

  • Aliquot upon receipt to avoid repeated freeze-thaw cycles

  • For short-term storage (1-2 weeks), 4°C is acceptable if protected from light

• Handling considerations:

  • Allow antibody to equilibrate to room temperature before opening vial

  • Centrifuge briefly before opening to collect liquid at the bottom

  • Avoid exposure to strong light during handling

  • Return to appropriate storage temperature immediately after use

• Buffer composition impact:

  • Standard storage buffer: "0.01 M PBS, pH 7.4, 0.03% Proclin-300 and 50% Glycerol"

  • The high glycerol concentration (50%) prevents freezing at -20°C

  • Proclin-300 serves as a preservative as noted: "This product contains ProClin: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only"

• Stability indicators:

  • Visible precipitation may indicate denaturation

  • Significant decrease in fluorescence intensity suggests FITC degradation

  • Unexplained increase in non-specific binding may indicate antibody degradation

• Working solution preparation:

  • Prepare fresh dilutions for each experiment

  • Use high-quality, filtered buffers

  • Add protein carriers (0.1-1% BSA) to diluted antibody to prevent adsorption to tubes

How can researchers address high background when using ECEL1 antibody, FITC conjugated?

High background is a common challenge with fluorescently labeled antibodies. Specific approaches include:

• Optimize blocking conditions:

  • Increase blocking time (2+ hours or overnight)

  • Try different blocking agents (BSA, normal serum, commercial blockers)

  • Include 0.1-0.3% Triton X-100 in blocking buffer for cell permeabilization

• Adjust antibody concentration:

  • Increase dilution (use less antibody) if background is uniformly high

  • Consider using the purified ECEL1 antibody (>95% protein G purified) to reduce non-specific binding

• Modify washing protocol:

  • Increase number of washes (5-6 times instead of 3)

  • Extend washing time (10-15 minutes per wash)

  • Add low concentration of detergent (0.05% Tween-20) to wash buffer

• Include additional controls:

  • Use FITC-conjugated isotype control (rabbit IgG-FITC) at same concentration

  • Include antigen pre-adsorption controls

  • Examine autofluorescence in unstained samples

• Tissue/sample-specific optimizations:

  • For tissues with high autofluorescence: Treat with Sudan Black B (0.1-0.3%)

  • For fixed tissues: Reduce fixation time or try different fixatives

  • For cells: Grow on appropriate substrates (collagen, poly-L-lysine)

How can researchers distinguish between specific and non-specific binding in ECEL1 antibody, FITC conjugated experiments?

Distinguishing specific from non-specific signals requires methodical validation:

• Essential controls:

  • Negative controls:

    • ECEL1-negative tissues or cells

    • Primary antibody omission

    • FITC-conjugated isotype control (rabbit IgG)

  • Positive controls:

    • Tissues with known ECEL1 expression (central nervous system)

    • ECEL1-overexpressing cell lines

• Pattern analysis:

  • Specific binding should match known ECEL1 localization (predominantly in the central nervous system)

  • Expected subcellular localization is consistent with a type II membrane protein

  • Non-specific binding often appears as:

    • Uniform staining across all cell types

    • Unusual subcellular patterns (e.g., nucleolar staining when target is membranous)

    • Edge or fold artifacts in tissue sections

• Signal verification techniques:

  • Peptide competition: Pre-incubate antibody with recombinant ECEL1 (AA 425-775) before staining

  • siRNA knockdown: Compare staining between ECEL1-knockdown and control cells

  • Dual labeling: Use a second ECEL1 antibody recognizing a different epitope

• Quantitative assessment:

  • Compare signal-to-noise ratios across different conditions

  • Use image analysis software to quantify staining intensity in regions of interest

  • Compare staining patterns with published literature on ECEL1 expression

What are common technical challenges when using FITC-conjugated antibodies and how can they be addressed?

FITC-conjugated antibodies present specific technical challenges:

• Photobleaching:

  • Issue: FITC photobleaches more rapidly than many other fluorophores

  • Solutions:

    • Minimize exposure to excitation light

    • Use anti-fade mounting media containing DABCO or similar compounds

    • Consider imaging FITC channels first in multi-fluorophore experiments

    • Reduce light intensity and increase detector sensitivity if possible

• pH sensitivity:

  • Issue: FITC fluorescence decreases significantly below pH 7.0

  • Solutions:

    • Ensure buffers are maintained at pH 7.2-7.4

    • Avoid acidic fixatives when possible

    • Use pH indicators in solutions to monitor changes

• Auto-fluorescence interference:

  • Issue: Cellular components (especially in fixed tissues) can auto-fluorescence in the FITC channel

  • Solutions:

    • Include unstained controls to assess autofluorescence

    • Use Sudan Black B treatment (0.1% in 70% ethanol) to reduce autofluorescence

    • Consider spectral unmixing during image acquisition if available

• Conjugation-related issues:

  • Issue: FITC conjugation might affect epitope binding

  • Solutions:

    • Compare results with unconjugated ECEL1 antibodies when possible

    • Validate with alternative detection methods (e.g., Western blot)

    • Consider using unconjugated primary with FITC-conjugated secondary if problems persist

How can ECEL1 antibody, FITC conjugated contribute to motor neuron development and neurodegenerative research?

ECEL1 plays critical roles in motor neuron development and potentially in neurodegeneration:

• Neuromuscular junction formation studies:

  • ECEL1 is essential for terminal arborization of motor axons

  • FITC-conjugated antibodies enable visualization of:

    • ECEL1 localization during developmental stages

    • Differences between wild-type and mutant protein distribution

    • Co-localization with other synaptic markers

• Axonal regeneration research:

  • "Mature Dine-deficient mice in which the lethality is rescued by genetic manipulation have shown the involvement of DINE in central nervous system regeneration"

  • ECEL1 antibody, FITC conjugated can be used to:

    • Track ECEL1 expression changes after nerve injury

    • Monitor dynamic protein relocalization during regeneration

    • Identify cells upregulating ECEL1 in response to damage

• Distal arthrogryposis disease modeling:

  • Various ECEL1 mutations cause DA5D with distinct phenotypes

  • Research applications include:

    • Comparing wild-type and mutant ECEL1 trafficking in motor neurons

    • Assessing structural changes using antibody epitope accessibility

    • Screening potential therapeutic compounds that correct ECEL1 mutant localization

• Integration with advanced imaging techniques:

  • Super-resolution microscopy to visualize ECEL1 at the neuromuscular junction

  • Live-cell imaging to track ECEL1 dynamics during development

  • Correlative light and electron microscopy to link ECEL1 distribution with ultrastructural features

How can structural biology approaches enhance the application of ECEL1 antibody, FITC conjugated in research?

Structural biology insights can significantly enhance antibody applications:

• Structure-guided epitope mapping:

  • The ECEL1 antibody targets AA 425-775

  • 3D modeling of ECEL1 can predict epitope accessibility in different conformational states

  • As noted in search results: "The three-dimensional structure of the ECEL1 protein was generated with the method of the homology modeling through the Swiss model server using PDB ID: 3dwb as a template"

• Mutation impact prediction:

  • Molecular dynamics simulations reveal structural changes in ECEL1 mutants

  • One study demonstrated "remarkable constructional differences by MD simulation between wild‐type and novel mutant of ECEL1 gene"

  • These insights can help researchers:

    • Predict how mutations affect antibody binding

    • Design experiments targeting specific structural domains

    • Interpret unexpected staining patterns in patient samples

• Zinc binding domain analysis:

  • ECEL1 is a zinc metalloprotease

  • Research showed "the reason for the lack of the Zn ion binding in mutation in the ECEL1 protein"

  • FITC-conjugated antibodies can be used to:

    • Assess conformational changes upon zinc binding/chelation

    • Compare mutants with altered zinc binding capacity

    • Evaluate folding and trafficking of catalytically inactive variants

• Integration with protein modeling:

  • Ramachandran plots from PROCHECK showed "90.0% of residues lie in most favored regions, 9.3% in additional allowed regions, 0.3% in generously allowed regions, and 0.3% in disallowed regions"

  • This structural information helps researchers:

    • Design experiments targeting specific structural domains

    • Interpret antibody accessibility in different protein conformations

    • Develop structure-based hypotheses for ECEL1 function

What emerging technologies can be combined with ECEL1 antibody, FITC conjugated for advanced neuroscience research?

Several cutting-edge technologies can enhance ECEL1 research when combined with FITC-conjugated antibodies:

• i-shaped antibody engineering:

  • Recent research describes "i-shaped antibody engineering enables conformational tuning"

  • Potential applications with ECEL1 antibodies include:

    • Creating conformation-specific ECEL1 antibodies

    • Developing antibodies that distinguish between wild-type and mutant forms

    • Engineering higher-affinity variants for low-abundance detection

• Single-cell analysis techniques:

  • Flow cytometry can analyze ECEL1 expression in neuronal subpopulations

  • FITC-conjugated antibodies enable:

    • Cell sorting of ECEL1-positive populations for downstream analysis

    • Quantification of expression levels at single-cell resolution

    • Correlation of ECEL1 expression with other neuronal markers

• Advanced microscopy methods:

  • Super-resolution techniques overcome the diffraction limit

  • Applications with ECEL1 antibody, FITC conjugated:

    • Nanoscale localization of ECEL1 at the neuromuscular junction

    • Co-localization with synaptic markers at molecular resolution

    • Tracking dynamic redistribution during development or regeneration

• In vivo imaging adaptations:

  • Modified FITC-conjugated antibodies for in vivo applications

  • Potential approaches:

    • Coupling with tissue clearing techniques for whole-organ imaging

    • Adaptation for intravital microscopy in animal models

    • Development of smaller antibody fragments with enhanced tissue penetration

• Organoid and 3D culture systems:

  • Neural organoids model development and disease

  • ECEL1 antibody, FITC conjugated enables:

    • 3D visualization of ECEL1 distribution in developing neural tissues

    • Comparison between control and DA5D patient-derived organoids

    • Assessment of therapeutic interventions targeting ECEL1 function