Etfdh Antibody

What is ETFDH and why is it important in metabolic research?

ETFDH (electron-transferring-flavoprotein dehydrogenase) is a crucial 617 amino acid membrane-bound electron transfer protein localized to the inner mitochondrial membrane. It plays a vital role in mitochondrial metabolism by accepting electrons from electron-transfer flavoprotein (ETF) in the mitochondrial matrix and subsequently reducing ubiquinone in the mitochondrial membrane . This process is essential for efficient energy production and metabolic regulation. ETFDH contains one molecule of flavin adenine dinucleotide (FAD) and a 4Fe-4S cluster, which are critical for electron transfer activity . The protein is particularly significant in research because mutations in the ETFDH gene have been linked to multiple acyl-CoA dehydrogenation deficiency (MADD) and glutaric aciduria type 2C (GA2C), characterized by severe metabolic disturbances including hypoketotic hypoglycemia and acidosis .

What types of ETFDH antibodies are available for research applications?

Several types of ETFDH antibodies are available for research, each with distinct characteristics:

This diversity allows researchers to select the most appropriate antibody based on their specific experimental needs, target species, and application requirements.

How do I select the appropriate ETFDH antibody for my specific research application?

Selecting the appropriate ETFDH antibody requires consideration of several factors:

• Experimental application: Determine whether you need the antibody for Western blotting, immunohistochemistry, immunofluorescence, flow cytometry, or immunoprecipitation. For example, if performing Western blotting, antibody 11109-1-AP has demonstrated efficacy across multiple species with a dilution range of 1:1000-1:6000 .

• Species reactivity: Match the antibody to your experimental model. For instance, if working with human samples only, ab103910 is suitable, while ab131376 offers broader reactivity across human, mouse, and rat samples .

• Antibody type: Consider whether a monoclonal or polyclonal antibody better suits your needs. Monoclonal antibodies like ab131376 offer high specificity to a single epitope, while polyclonal antibodies like CAB6585 recognize multiple epitopes, potentially providing stronger signals but with possible increased background .

• Validation data: Review the available validation data for each antibody. For example, sc-515202 has been validated for multiple applications including Western blotting, immunoprecipitation, and immunofluorescence .

• Target location: Consider the subcellular localization of ETFDH (inner mitochondrial membrane) and ensure the antibody's epitope is accessible in your experimental conditions.

What are the optimal conditions for Western blot analysis using ETFDH antibodies?

For optimal Western blot analysis using ETFDH antibodies, follow these methodological guidelines:

• Sample preparation:

  • For tissue homogenates, use 10-15 μg of protein as demonstrated with human liver, heart, and various other tissue homogenates .

  • For cell lysates, load 15 μg of protein as shown effective with HepG2, HeLa, HDFn, SY5Y, and Jurkat cell lines .

• Electrophoresis and transfer:

  • Use 10% SDS-PAGE for optimal protein separation .

  • Transfer to PVDF membranes for better protein retention and antibody binding .

• Blocking and antibody incubation:

  • Block with 5% non-fat milk in TBST at room temperature for 1 hour .

  • Use recommended antibody dilutions: 1:1000-1:6000 for 11109-1-AP , 1 μg/mL for ab131376 , or 1 μg/mL for ab103910 .

  • Incubate with primary antibody overnight at 4°C for optimal binding .

• Detection and visualization:

  • Use appropriate secondary antibodies, such as goat anti-mouse AP at 1/3000 dilution for ab131376 .

  • Develop using ECL detection systems for sensitive protein detection .

• Band interpretation:

  • Expect the ETFDH protein band at approximately 68-70 kDa .

  • Quantify band intensity using software like Quantity One or ImageJ .

How should I optimize immunohistochemistry protocols for ETFDH detection in tissue samples?

For optimized immunohistochemistry protocols with ETFDH antibodies:

• Tissue fixation and processing:

  • Use formalin-fixed, paraffin-embedded tissue sections for consistent results .

  • Perform heat-mediated antigen retrieval using either:

    • Sodium citrate buffer (pH 6.0) for epitope retrieval , or

    • TE buffer (pH 9.0) as suggested for antibody 11109-1-AP .

• Antibody concentration and incubation:

  • For ab131376, use at 5 μg/ml concentration .

  • For 11109-1-AP, use at 1:50-1:500 dilution .

  • Incubate sections with primary antibody for 15 minutes to 2 hours at room temperature, or overnight at 4°C for weaker signals .

• Detection systems:

  • Use HRP-conjugated compact polymer systems for sensitive detection .

  • Develop with DAB as the chromogen and counterstain with hematoxylin .

• Controls and validation:

  • Include positive control tissues known to express ETFDH (liver, heart, kidney) .

  • Use isotype controls to assess non-specific binding.

  • Be aware that high background signal may occur in mouse tissue due to direct reaction between mouse IgG in tissue and anti-mouse secondary antibodies .

• Interpretation:

  • Look for mitochondrial localization pattern consistent with ETFDH's function.

  • Assess staining intensity and distribution patterns in relation to known ETFDH expression in different tissues.

What are the recommended procedures for immunofluorescence detection of ETFDH?

For effective immunofluorescence detection of ETFDH:

• Cell preparation and fixation:

  • Fix cells with 4% paraformaldehyde for 20 minutes .

  • Permeabilize with 0.1% Triton X-100 for 15 minutes to allow antibody access to mitochondrial targets .

• Blocking and antibody incubation:

  • Block with 1% BSA for all blocking steps to reduce background .

  • Incubate with ETFDH antibody (e.g., ab131376) at 5 μg/ml for 2 hours at room temperature or overnight at 4°C .

  • Use fluorophore-conjugated secondary antibody such as Alexa Fluor® 594 Goat anti-Mouse IgG at 1/1000 dilution .

• Co-localization studies:

  • Consider dual labeling with mitochondrial markers to confirm subcellular localization.

  • Use confocal microscopy for precise localization analysis.

• Image acquisition and analysis:

  • Capture images using appropriate filters for the selected fluorophore.

  • Analyze mitochondrial distribution patterns typical of ETFDH localization.

  • Quantify signal intensity using specialized image analysis software.

How can ETFDH antibodies be used to investigate mitochondrial dysfunction in metabolic disorders?

ETFDH antibodies can be strategically employed to investigate mitochondrial dysfunction through multiple approaches:

• Expression level analysis:

  • Utilize Western blotting with ETFDH antibodies to quantify protein expression levels in patient samples versus controls .

  • Compare ETFDH protein levels across different tissues to map metabolic dysfunction patterns, particularly in tissues with high energy demands like liver, heart, and skeletal muscle .

• Tissue distribution studies:

  • Perform immunohistochemistry with antibodies like 11109-1-AP to examine ETFDH distribution in normal versus diseased tissues .

  • Compare staining patterns between affected and unaffected tissues to identify localized metabolic disruptions.

• Functional interaction studies:

  • Use immunoprecipitation with antibodies like sc-515202 or 11109-1-AP to isolate ETFDH and analyze its interaction partners .

  • Investigate potential alterations in ETFDH-ETF complex formation in disease states.

• Genetic-phenotypic correlation:

  • Combine genetic analysis of ETFDH mutations with antibody-based protein studies to establish genotype-phenotype correlations.

  • Use siRNA knockdown of ETFDH followed by antibody detection to model the effects of gene mutations on protein expression and function .

• Therapeutic response monitoring:

  • Apply ETFDH antibodies to monitor protein expression changes in response to treatments, particularly in riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency (RR-MADD) .

What approaches can be used to validate ETFDH antibody specificity for critical research applications?

To validate ETFDH antibody specificity for high-confidence research applications:

• Genetic validation approaches:

  • Perform siRNA knockdown of ETFDH and verify reduced antibody signal by Western blot, as demonstrated in the HEK293T cell model .

  • Use CRISPR/Cas9-mediated knockout models to confirm complete loss of antibody signal.

• Multiple antibody concordance:

  • Compare results using different ETFDH antibodies targeting distinct epitopes (e.g., ab131376 vs. CAB6585) .

  • Verify consistent detection patterns across monoclonal and polyclonal antibodies like ab131376 (monoclonal) and 11109-1-AP (polyclonal) .

• Recombinant protein controls:

  • Use purified recombinant ETFDH protein as a positive control.

  • Perform peptide competition assays to confirm epitope specificity.

• Cross-species reactivity assessment:

  • Test antibody reactivity across human, mouse, and rat samples to verify evolutionary conservation of the recognized epitope .

  • Compare detection patterns in different species with known ETFDH sequence homology.

• Immunoprecipitation-mass spectrometry validation:

  • Perform IP with ETFDH antibody followed by mass spectrometry to confirm the identity of the pulled-down protein.

  • Verify identification of expected peptides from the ETFDH sequence.

How can ETFDH antibodies be incorporated into multiparametric flow cytometry for analyzing metabolic alterations?

For incorporating ETFDH antibodies into multiparametric flow cytometry:

• Cell preparation and fixation:

  • Fix cells with methanol for optimal permeabilization of mitochondrial membranes .

  • Ensure thorough permeabilization to allow antibody access to the inner mitochondrial membrane where ETFDH is located.

• Antibody selection and staining:

  • Use flow cytometry-validated antibodies like ab131376 at 1 μg/mL concentration .

  • Include isotype control antibodies to establish background staining levels.

  • Block with 1% BSA to reduce non-specific binding .

• Multiparametric panel design:

  • Combine ETFDH staining with other metabolic markers such as:

    • Mitochondrial membrane potential dyes (TMRM, JC-1)

    • Reactive oxygen species indicators (MitoSOX, CellROX)

    • Additional mitochondrial protein markers (Complex I-V components)

  • Include viability dyes to exclude dead cells from analysis.

• Gating strategy and analysis:

  • First gate on intact cells using forward and side scatter.

  • Apply viability gating to exclude dead cells.

  • Analyze ETFDH expression levels relative to other mitochondrial parameters.

  • Use median fluorescence intensity (MFI) for quantitative comparisons between experimental groups.

• Experimental applications:

  • Compare ETFDH expression between normal and patient-derived cells.

  • Assess correlation between ETFDH levels and functional mitochondrial parameters.

  • Monitor changes in ETFDH expression following metabolic challenges or treatments.

How can I address high background issues when using ETFDH antibodies in immunohistochemistry?

To address high background issues in ETFDH immunohistochemistry:

• Species cross-reactivity concerns:

  • Be aware that high background signal in mouse tissue samples may result from direct reaction between mouse IgG in tissue and goat anti-mouse secondary antibodies .

  • For mouse tissues, consider using mouse-on-mouse detection systems or primary antibodies from different host species (rabbit or goat).

• Optimization strategies:

  • Titrate antibody concentration – test dilution series (e.g., 1:50, 1:100, 1:200, 1:500) to identify optimal signal-to-noise ratio .

  • Modify blocking conditions – increase BSA concentration to 2-5% or add 5-10% normal serum from the same species as the secondary antibody.

  • Extend blocking time to 2 hours at room temperature.

  • Increase washing stringency with additional wash steps or higher detergent concentration.

• Antigen retrieval modifications:

  • Compare citrate buffer (pH 6.0) versus TE buffer (pH 9.0) for optimal epitope retrieval .

  • Adjust retrieval time and temperature to optimize signal specificity.

• Detection system alternatives:

  • Switch between polymer-based and avidin-biotin systems to identify optimal detection method.

  • Consider tyramide signal amplification for weak signals while maintaining low background.

• Control implementations:

  • Use isotype controls at equivalent concentrations to primary antibody.

  • Include no-primary antibody controls to identify secondary antibody non-specific binding.

  • Use tissues with known negative expression of ETFDH as negative controls.

What strategies can resolve inconsistent Western blot results with ETFDH antibodies?

For resolving inconsistent Western blot results with ETFDH antibodies:

• Sample preparation considerations:

  • Ensure complete protein denaturation by heating samples at 95°C for 5 minutes in presence of reducing agents.

  • Use mitochondria-specific extraction buffers to enrich for ETFDH protein.

  • Add protease inhibitors to prevent degradation, especially important for mitochondrial membrane proteins.

• Electrophoresis and transfer optimization:

  • Adjust polyacrylamide percentage (8-12%) for optimal separation near the expected 68-70 kDa range .

  • Modify transfer conditions (time, voltage, buffer composition) for efficient transfer of mitochondrial membrane proteins.

  • Consider using PVDF membranes instead of nitrocellulose for better protein retention .

• Antibody selection and dilution:

  • Compare different antibodies targeting distinct ETFDH epitopes:

    • Try polyclonal antibodies like 11109-1-AP (1:1000-1:6000) for higher sensitivity .

    • Use monoclonal antibodies like ab131376 (1 μg/mL) for higher specificity .

  • Perform time-course incubations to determine optimal binding conditions.

• Signal development strategies:

  • For weak signals, extend exposure time or switch to more sensitive ECL substrates.

  • For multiple bands or high background, increase washing stringency and optimize blocking conditions.

  • Consider using different secondary antibodies or detection systems.

• Control implementations:

  • Include positive control samples from tissues with high ETFDH expression (liver, heart, kidney) .

  • Use recombinant ETFDH protein as a standard reference.

  • Implement loading controls appropriate for mitochondrial proteins.

How do I interpret and validate unexpected ETFDH antibody binding patterns in disease models?

For interpreting and validating unexpected ETFDH antibody binding patterns:

• Pattern verification approaches:

  • Confirm unexpected patterns using multiple ETFDH antibodies targeting different epitopes .

  • Verify at both protein level (Western blot) and cellular level (immunohistochemistry/immunofluorescence).

  • Compare results across different experimental conditions and tissue/cell types.

• Molecular validation strategies:

  • Correlate protein observations with mRNA expression using RT-PCR .

  • Perform minigene splice assays to investigate potential alternative splicing or mutation effects on ETFDH .

  • Sequence the ETFDH gene in the disease model to identify potential mutations that might affect antibody binding.

• Functional correlation analysis:

  • Relate observed binding patterns to functional mitochondrial assays.

  • Investigate whether unexpected patterns correlate with metabolic phenotypes.

  • Compare patterns with biochemical indicators of ETFDH dysfunction.

• Alternative isoform considerations:

  • Investigate whether unexpected patterns might represent detection of alternative ETFDH splice variants .

  • Check antibody epitope location against known alternative splicing regions.

  • Perform isoform-specific PCR to verify expression of alternative transcripts.

• Disease-specific modifications:

  • Consider post-translational modifications that might occur in disease states.

  • Investigate potential protein degradation patterns specific to the disease model.

  • Examine protein-protein interactions that might mask or alter epitope accessibility.

How can ETFDH antibodies be used to investigate pathogenic mechanisms in fatty acid oxidation disorders?

ETFDH antibodies provide powerful tools for investigating pathogenic mechanisms in fatty acid oxidation disorders through several methodological approaches:

• Patient sample analysis:

  • Compare ETFDH protein levels and localization in control versus patient samples using Western blot, IHC, or IF .

  • Correlate ETFDH expression levels with clinical severity and metabolic biomarkers.

  • Analyze tissue-specific expression patterns, particularly in affected tissues like muscle, liver, and heart .

• Mutation impact assessment:

  • Use ETFDH antibodies to assess protein expression levels in cells with identified ETFDH mutations.

  • Investigate how specific mutations (e.g., c.487+2T>A) affect protein stability and expression through Western blot analysis .

  • Combine with minigene splice assays to correlate splicing defects with protein expression patterns .

• NMD pathway investigation:

  • Use siRNA approaches to knockdown ETFDH followed by antibody detection to model nonsense-mediated decay mechanisms .

  • Compare protein degradation rates between wild-type and mutant ETFDH.

  • Assess how NMD inhibitors affect protein levels of mutant ETFDH variants.

• Therapeutic response monitoring:

  • Apply antibodies to track ETFDH protein restoration in riboflavin-responsive MADD following treatment.

  • Monitor changes in subcellular localization and expression levels in response to therapeutic interventions.

  • Correlate protein level changes with metabolic and clinical improvements.

• Protein interaction studies:

  • Use immunoprecipitation with antibodies like 11109-1-AP or sc-515202 to investigate altered protein interactions in disease states .

  • Assess whether mutations affect ETFDH interaction with electron transfer flavoprotein (ETF) or other components of the respiratory chain.

What role do ETFDH antibodies play in studying the relationship between mitochondrial dysfunction and neurodegenerative diseases?

ETFDH antibodies enable detailed investigation of mitochondrial dysfunction in neurodegenerative contexts:

• Expression profiling in neural tissues:

  • Use antibodies like ab131376 or 11109-1-AP to compare ETFDH expression in normal versus neurodegenerative brain tissues .

  • Perform region-specific analysis of brain areas selectively affected in different neurodegenerative diseases.

  • Analyze expression in different neural cell types (neurons, astrocytes, microglia) through co-localization immunofluorescence studies.

• Oxidative stress correlation:

  • Combine ETFDH immunodetection with oxidative stress markers to establish relationships between electron transport chain dysfunction and oxidative damage.

  • Assess whether ETFDH expression or localization changes precede or follow oxidative damage in disease models.

• Mitochondrial dynamics investigation:

  • Use ETFDH antibodies alongside markers of mitochondrial fission/fusion to examine relationships between electron transport and mitochondrial morphology.

  • Analyze whether ETFDH distribution changes with altered mitochondrial network structure in neurodegenerative conditions.

• Metabolic reprogramming assessment:

  • Investigate how ETFDH expression correlates with metabolic shifts in neurodegenerative disease models.

  • Combine with metabolomic approaches to relate ETFDH changes to specific metabolic pathway alterations.

• Therapeutic target identification:

  • Use antibodies to screen compounds that might stabilize or upregulate ETFDH expression.

  • Monitor ETFDH levels in response to potential neuroprotective interventions targeting mitochondrial function.

How can ETFDH antibody-based techniques be integrated with genetic analysis in precision medicine approaches?

Integrating ETFDH antibody techniques with genetic analysis creates powerful precision medicine applications:

• Genotype-phenotype correlation studies:

  • Combine genetic sequencing of ETFDH mutations with antibody-based protein expression analysis .

  • Use antibodies to determine how specific genetic variants affect protein expression, stability, and localization.

  • Develop predictive models relating genetic variants to protein expression patterns and disease severity.

• Variant functional validation:

  • Apply antibodies to verify the functional impact of variants of uncertain significance (VUS) identified through genetic testing.

  • Use cell models expressing patient-specific variants to assess protein expression and function.

  • Compare expression levels of wild-type versus mutant ETFDH protein in patient-derived cells.

• Personalized treatment monitoring:

  • Track ETFDH protein levels in patient samples before and after therapeutic interventions.

  • Correlate changes in protein expression with clinical improvements.

  • Use antibody-based assays as biomarkers for treatment response.

• Multi-omics integration:

  • Combine antibody-detected protein expression data with:

    • Transcriptomic data on ETFDH mRNA expression

    • Metabolomic profiles of acylcarnitines and other relevant metabolites

    • Clinical phenotyping data

  • Develop integrated models predicting disease progression and treatment response.

• Screening strategies for at-risk populations:

  • Develop antibody-based screening approaches to complement genetic testing.

  • Validate protein-level biomarkers that might indicate ETFDH dysfunction even in the absence of known pathogenic variants.

  • Create diagnostic algorithms integrating genetic and protein-level data.

How might single-cell analysis using ETFDH antibodies advance our understanding of metabolic heterogeneity?

Single-cell analysis with ETFDH antibodies presents transformative potential for understanding metabolic heterogeneity:

• Single-cell immunofluorescence techniques:

  • Apply antibodies like ab131376 in high-resolution imaging to assess cell-to-cell variation in ETFDH expression .

  • Implement automated image analysis to quantify expression levels across thousands of individual cells.

  • Correlate ETFDH expression with morphological features and other cellular parameters.

• Mass cytometry (CyTOF) applications:

  • Develop metal-conjugated ETFDH antibodies for high-dimensional protein profiling.

  • Create panels combining ETFDH with other metabolic enzymes, mitochondrial markers, and cell type identifiers.

  • Map metabolic phenotypes across heterogeneous tissue populations.

• Single-cell Western approaches:

  • Adapt antibodies like 11109-1-AP (1:1000-1:6000) for microfluidic single-cell Western blotting .

  • Quantify ETFDH protein levels in individual cells to assess population heterogeneity.

  • Compare expression variance in normal versus disease states.

• Integration with single-cell genomics/transcriptomics:

  • Develop protocols combining ETFDH protein detection with single-cell RNA sequencing.

  • Correlate protein expression with transcript levels at single-cell resolution.

  • Identify regulatory factors controlling ETFDH expression heterogeneity.

• Spatial metabolomics correlation:

  • Combine ETFDH immunohistochemistry with spatial metabolomics to relate protein expression to local metabolite concentrations.

  • Map metabolic microenvironments in tissues with heterogeneous ETFDH expression.

  • Develop predictive models of how cellular ETFDH expression influences local metabolism.

What novel biomarker approaches might employ ETFDH antibodies for early detection of mitochondrial dysfunction?

Innovative biomarker approaches utilizing ETFDH antibodies include:

• Liquid biopsy applications:

  • Develop sensitive ELISA assays using antibodies like 11109-1-AP or sc-515202 to detect circulating ETFDH in blood samples .

  • Investigate ETFDH in extracellular vesicles as potential biomarkers of mitochondrial stress.

  • Correlate circulating ETFDH levels with tissue dysfunction in mitochondrial disorders.

• Multiplex antibody arrays:

  • Create protein arrays incorporating ETFDH antibodies alongside other mitochondrial markers.

  • Develop signature profiles of mitochondrial dysfunction across multiple proteins.

  • Implement machine learning approaches to identify patterns predictive of disease states.

• Post-translational modification mapping:

  • Develop modification-specific antibodies detecting phosphorylated, acetylated, or otherwise modified ETFDH.

  • Investigate whether specific modifications serve as early indicators of mitochondrial stress.

  • Track changes in modification patterns during disease progression.

• Imaging biomarker development:

  • Adapt antibodies for in vivo molecular imaging applications.

  • Develop minimally invasive approaches to monitor ETFDH in accessible tissues (skin, blood cells).

  • Create correlation models between peripheral ETFDH expression and central organ function.

• High-throughput screening platforms:

  • Implement antibody-based assays in automated platforms for population screening.

  • Develop risk stratification algorithms based on ETFDH expression patterns.

  • Create POC (point-of-care) testing approaches for rapid assessment of mitochondrial health.

How might advances in antibody engineering enhance future ETFDH research applications?

Emerging antibody engineering approaches will expand ETFDH research capabilities:

• Nanobody and single-domain antibody development:

  • Engineer smaller antibody formats for improved penetration into mitochondrial structures.

  • Develop intrabodies that can track ETFDH in living cells without interfering with function.

  • Create bivalent constructs recognizing ETFDH and interacting partners simultaneously.

• Engineered antibody fragments:

  • Design Fab and scFv fragments with enhanced tissue penetration.

  • Develop constructs optimized for super-resolution microscopy applications.

  • Create antibody fragments capable of passing through biological barriers for in vivo applications.

• Bifunctional antibody applications:

  • Engineer ETFDH antibody constructs fused to:

    • Proximity labeling enzymes for interactome mapping

    • Fluorescent proteins for real-time tracking

    • Mitochondrial targeting sequences for enhanced localization

  • Develop therapeutic constructs combining ETFDH targeting with functional domains.

• Conformation-specific antibodies:

  • Generate antibodies specifically recognizing active versus inactive ETFDH conformations.

  • Develop tools to detect misfolded ETFDH variants in disease states.

  • Create sensors of ETFDH functional status rather than just presence/absence.

• Humanized antibody development:

  • Engineer fully humanized ETFDH antibodies for potential therapeutic applications.

  • Develop constructs capable of stabilizing mutant ETFDH protein.

  • Create antibody-drug conjugates for targeted delivery to mitochondria.