EHHADH Antibody

What is EHHADH and why is it important in research?

EHHADH (Enoyl-CoA Hydratase And 3-Hydroxyacyl CoA Dehydrogenase) is a bifunctional enzyme and one of the four essential enzymes in the peroxisomal beta-oxidation pathway. This 723 amino acid protein contains an N-terminal region with enoyl-CoA hydratase activity and a C-terminal region with 3-hydroxyacyl-CoA dehydrogenase activity . It also possesses delta 3, delta 2-enoyl-CoA isomerase activity. EHHADH plays critical roles in:

• Catalyzing two of the four reactions in the long-chain fatty acids peroxisomal beta-oxidation pathway

• Breaking down branched-chain fatty acids

• Regulating medium-chain dicarboxylic fatty acids, which are essential regulators of all fatty acid oxidation pathways

• Contributing to the degradation of long-chain dicarboxylic acids through peroxisomal beta-oxidation

This multifunctionality makes EHHADH a significant research target in understanding peroxisomal disorders, metabolic diseases, and potentially cancer biology.

For optimal Western blot detection of EHHADH, follow these methodological steps:

• Sample preparation:

  • Lyse cells or tissues in RIPA buffer supplemented with protease and phosphatase inhibitors

  • Sonicate samples followed by centrifugation (10 min at 12,000 rpm at 4°C)

  • Determine protein concentration using the BCA method

• Gel electrophoresis and transfer:

  • Separate proteins on 4-12% Bis-Tris gels

  • Transfer onto nitrocellulose membrane

• Antibody incubation:

  • Primary antibody dilutions:

    • ab123490: 0.4 μg/mL

    • ab136059: 1/500 dilution

    • 26570-1-AP: 1:1000-1:6000

  • Secondary antibody: Anti-rabbit IgG with appropriate detection system

• Detection:

  • Visualize using enhanced chemiluminescence (ECL) or infrared imaging systems

  • Expected molecular weight: 79 kDa (predicted), though observed at 69 kDa in some systems

• Controls:

  • Positive controls: HeLa, 293T, Jurkat, SMMC-7721, and HepG2 cells; mouse/rat kidney and liver tissues

How should EHHADH antibodies be stored for optimal stability?

For maximum stability and performance of EHHADH antibodies, follow these specific storage recommendations:

• Store at -20°C for long-term stability

• Store in PBS buffer containing 0.02% sodium azide and 50% glycerol pH 7.3

• Products are typically stable for one year after shipment when stored correctly

• Aliquoting is generally unnecessary for -20°C storage

• Some antibody preparations (e.g., 20μl sizes) may contain 0.1% BSA for added stability

Avoid repeated freeze-thaw cycles which can significantly reduce antibody performance. For working solutions, store at 4°C for short periods (up to one week) but return to -20°C for longer storage intervals.

How can EHHADH antibodies be used to investigate peroxisomal disorders?

EHHADH antibodies provide valuable tools for investigating peroxisomal disorders through multiple methodological approaches:

• Expression analysis in disease models:

  • Western blot and immunohistochemistry can detect alterations in EHHADH expression levels in patient samples or disease models

  • Defects in EHHADH have been linked to peroxisomal disorders such as Zellweger syndrome

• Interaction studies with other peroxisomal proteins:

  • Co-immunoprecipitation using EHHADH antibodies can identify interaction partners

  • EHHADH works with HSD17B4 to catalyze the hydration of trans-2-enoyl-CoA and the dehydrogenation of 3-hydroxyacyl-CoA, but with opposite chiral specificity

• Subcellular localization studies:

  • Immunofluorescence using anti-EHHADH antibodies can detect altered peroxisomal localization

  • The C-terminal tripeptide sequence is essential for peroxisomal transport, and mislocalization may indicate disorder

• Knockout/knockdown functional analysis:

  • EHHADH-null mice exhibit a blunted peroxisome proliferative response when challenged with peroxisome proliferators

  • This suggests that enoyl-CoAs may be diverted to the D-hydroxy-specific beta-oxidation system for metabolism

The combined application of these techniques with EHHADH antibodies enables comprehensive investigation of peroxisomal structure, function, and pathology in various disorders.

What is the significance of EHHADH in cancer research and how can antibodies aid investigation?

Recent research has implicated EHHADH in cancer biology, particularly in osteosarcoma (OS), where EHHADH antibodies can facilitate several investigative approaches:

These methodological approaches using EHHADH antibodies provide valuable insights into the role of this enzyme in cancer development and progression, potentially identifying new therapeutic targets.

How can researchers validate EHHADH antibody specificity for their experimental systems?

Rigorous validation of EHHADH antibody specificity is crucial for experimental reliability. Researchers should employ multiple approaches:

• Genetic knockdown/knockout validation:

  • Compare antibody reactivity in wild-type versus EHHADH knockdown or knockout samples

  • Example: EHHADH-null mice or cells treated with EHHADH siRNA should show significantly reduced or absent signal

• Overexpression systems:

  • Transfect cells with EHHADH expression plasmids (e.g., MGC premier cDNA clone for EHHADH, pCS6)

  • Confirm increased antibody signal in transfected versus non-transfected cells

• Positive and negative control tissues/cells:

  • Validated positive controls: HepG2 cells, SMMC-7721 cells, mouse/rat kidney and liver tissues

  • Use tissues known to have low EHHADH expression as negative controls

• Molecular weight verification:

  • Confirm detection at the predicted molecular weight (79 kDa) or the observed molecular weight (69 kDa in some systems)

  • Use appropriate molecular weight markers

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide before application

  • Specific antibody signal should be significantly reduced or eliminated

By applying these methodological approaches, researchers can confidently establish the specificity of their EHHADH antibodies for their particular experimental systems.

What are the key considerations when using EHHADH antibodies in studies of metabolic diseases?

When investigating metabolic diseases using EHHADH antibodies, researchers should consider these methodological aspects:

• Tissue-specific expression patterns:

  • EHHADH expression varies across tissues, with high expression in liver and kidney

  • Hepatic EHHADH expression has been negatively correlated with fasting plasma glucose levels in mouse models of diabetes

• Disease-specific alterations:

  • In diabetic kidney disease, endogenous EHHADH levels strongly correlate with progression and severity of nephropathy in T2D patients

  • EHHADH knockout mice exhibit worsened renal tubular injury in diabetic models

• Peroxisomal dynamics assessment:

  • EHHADH functions as a modulator of pexophagy (selective autophagy of peroxisomes)

  • In renal tubular epithelial cells, EHHADH knockdown induces dramatic loss of peroxisomes

  • This loss can be restored by autophagic inhibitors (3-methyladenine or bafilomycin A1)

• Oxidative stress correlation:

  • EHHADH deficiency increases reactive oxygen species (ROS) levels

  • ROS inhibition blocks pexophagy in EHHADH-deficient cells

• Coexpression network analysis:

  • EHHADH is part of a coexpression network with other metabolic genes

  • Consider analyzing expression patterns of these network genes (e.g., using antibodies against multiple targets) for comprehensive insights

These considerations enable researchers to design robust experiments that accurately assess EHHADH's role in metabolic disease pathogenesis and progression.

How can immunoprecipitation protocols be optimized for EHHADH studies?

Optimizing immunoprecipitation (IP) for EHHADH requires attention to specific methodological details:

• Optimal antibody selection:

  • Use antibodies specifically validated for IP, such as ab123490

  • Recommended concentration: 6 μg antibody per mg of lysate

• Lysate preparation:

  • Starting material: 1 mg of total protein lysate is recommended

  • Use freshly prepared lysates in a gentle lysis buffer to preserve protein-protein interactions

• Negative controls:

  • Include parallel IP with control IgG from the same species as the EHHADH antibody

  • This controls for non-specific binding

• Detection antibody considerations:

  • For Western blot detection after IP, use 1 μg/ml of anti-EHHADH (ab123490)

  • If using the same antibody for IP and detection, use a detection system that minimizes background from heavy and light chains

• Validation approach:

  • Compare IP results with antibodies recognizing different EHHADH epitopes

  • Example: IP with an antibody recognizing an upstream epitope followed by detection with ab123490

• Expected results:

  • Successful IP should yield a band at approximately 79 kDa

  • Validate with chemiluminescence detection with moderate exposure times (3 minutes has been reported as effective)

Following these optimized protocols will maximize EHHADH immunoprecipitation efficiency and specificity for interaction studies.

What role does EHHADH play in fatty acid metabolism research and which antibodies are most suitable?

EHHADH has emerged as a critical enzyme in specialized fatty acid metabolism pathways, particularly in the synthesis of docosahexaenoic acid (DHA):

• Role in the "Sprecher" pathway:

  • EHHADH has been demonstrated as essential for DHA synthesis

  • Overexpression of EHHADH promotes DHA synthesis

  • This provides a novel target for understanding omega-3 fatty acid metabolism

• Peroxisomal β-oxidation function:

  • EHHADH catalyzes two of the four reactions in the peroxisomal beta-oxidation pathway

  • It specifically functions in the L-specific branch of peroxisomal fatty acid oxidation

  • Works collaboratively with HSD17B4 but with opposite chiral specificity

• Recommended antibodies for metabolism studies:

  • For detection of endogenous EHHADH in metabolic tissues: 26570-1-AP (detects mouse/rat/human)

  • For co-immunoprecipitation with metabolic partners: ab123490

  • For tissue expression studies: 26570-1-AP for IHC applications at 1:100-1:400 dilution

• Methodological approach for metabolic pathway analysis:

  • Combine antibody detection with qRT-PCR for comprehensive analysis

  • For EHHADH knockout/overexpression studies, verify both mRNA and protein levels

  • Example primers for qRT-PCR: forward 5′-ATGGCTGAGTATCTGAGGCTG-3′ and reverse 5′-ACCGTATGGTCCAAACTAGCTT-3′

These insights and methodological approaches position EHHADH antibodies as valuable tools in understanding specialized fatty acid metabolism and related disorders.

What are common challenges in EHHADH antibody applications and how can they be resolved?

Researchers working with EHHADH antibodies may encounter several technical challenges that can be systematically addressed:

• Variable detection in different tissues:

  • Challenge: Inconsistent signal strength across tissue types

  • Solution: Optimize protein extraction based on tissue type; for highly fibrous tissues, use stronger lysis buffers with mechanical disruption methods

  • Validated positive controls: mouse kidney tissue, SMMC-7721 cells, HepG2 cells, mouse liver tissue, rat kidney tissue

• High background in immunohistochemistry:

  • Challenge: Non-specific staining obscuring specific EHHADH signal

  • Solution: Use antigen retrieval with TE buffer pH 9.0 or alternatively with citrate buffer pH 6.0

  • Optimal dilution: 1:100-1:400 for IHC applications

• Multiple bands in Western blot:

  • Challenge: Detection of unexpected bands besides the predicted 79 kDa band

  • Solution: Verify with knockout/knockdown controls; some antibodies detect at 69 kDa rather than the predicted 79 kDa

  • Use freshly prepared samples and include protease inhibitors to prevent degradation

• Weak signal in immunofluorescence:

  • Challenge: Poor signal-to-noise ratio in IF applications

  • Solution: Use higher antibody concentration (1:20-1:200) for immunofluorescence

  • Validated cell line: HepG2 cells show reliable detection in IF applications

These methodological adjustments can significantly improve the reliability and specificity of EHHADH antibody applications across different experimental contexts.

How can EHHADH antibodies be utilized in studying the role of peroxisomes in disease models?

EHHADH antibodies offer powerful tools for investigating peroxisomal involvement in disease pathogenesis through several methodological approaches:

• Peroxisomal proliferation assessment:

  • EHHADH-null mice exhibit blunted peroxisome proliferative responses to peroxisome proliferators

  • Monitor peroxisomal numbers using EHHADH antibodies in combination with other peroxisomal markers (e.g., Abcd3/PMP70)

• Pexophagy monitoring in disease models:

  • EHHADH functions as a modulator of pexophagy (selective autophagy of peroxisomes)

  • In diabetic kidney disease models, EHHADH deficiency accelerates renal injury through enhanced pexophagy

  • Use EHHADH antibodies in combination with autophagy markers (e.g., NBR1) to track this process

• Multi-protein complex analysis:

  • EHHADH works in concert with other peroxisomal proteins

  • Use co-immunoprecipitation with EHHADH antibodies followed by immunoblotting for interaction partners

  • Example partners include HSD17B4, which catalyzes similar reactions but with opposite chiral specificity

• ROS correlation studies:

  • EHHADH deficiency increases reactive oxygen species (ROS) levels

  • Combine EHHADH immunostaining with ROS detection methods to correlate peroxisomal function with oxidative stress

  • This is particularly relevant in diabetic nephropathy models where EHHADH deficiency worsens injury

These methodological approaches using EHHADH antibodies enable comprehensive investigation of peroxisomal dynamics in various disease models, providing mechanistic insights into pathogenesis.

What are the considerations for using EHHADH antibodies in co-localization studies with other peroxisomal proteins?

When conducting co-localization studies involving EHHADH and other peroxisomal proteins, researchers should address these methodological considerations:

• Antibody compatibility:

  • Select antibodies raised in different host species to allow simultaneous detection

  • Example combinations from the literature:

    • Anti-EHHADH (rabbit) with anti-Cyp4a10 (goat)

    • Anti-EHHADH (rabbit) with anti-Abcd3/PMP70 (different rabbit clone but using sequential staining)

• Fixation and permeabilization optimization:

  • Peroxisomal membranes may require specific permeabilization conditions

  • For co-localization of EHHADH with membrane proteins, test different detergents (Triton X-100, digitonin, or saponin) at various concentrations

• Resolution considerations:

  • Peroxisomes are small organelles (0.1-1 μm); super-resolution microscopy techniques may be necessary

  • Confocal microscopy with deconvolution is the minimum recommended for accurate co-localization analysis

• Controls for specificity:

  • Include single-antibody controls to verify lack of cross-reactivity

  • Use EHHADH knockout cells as negative controls

  • Include peroxisomal marker proteins (e.g., Abcd3/PMP70) as positive controls for peroxisomal localization

• Quantification methods:

  • Apply appropriate co-localization coefficients (Pearson's, Manders' coefficients)

  • Ensure sufficient number of cells are analyzed for statistical validity

  • Consider 3D analysis rather than single confocal sections for complete assessment

Following these methodological guidelines will enhance the reliability of co-localization studies involving EHHADH and other peroxisomal proteins.

How can EHHADH antibodies contribute to understanding the connection between metabolism and cancer?

EHHADH antibodies provide valuable tools for investigating the emerging links between peroxisomal metabolism and cancer through several methodological approaches:

These methodological approaches using EHHADH antibodies can reveal novel connections between peroxisomal metabolism and cancer biology, potentially identifying new therapeutic targets.

What emerging areas of EHHADH research might require specialized antibody applications?

Several cutting-edge research areas are highlighting new roles for EHHADH that will require specialized antibody applications:

• EHHADH in pexophagy regulation:

  • Recent research identifies EHHADH as a key modulator of pexophagy

  • Future studies will require antibodies optimized for tracking EHHADH during autophagic processes

  • Combined use with NBR1 (a pexophagy receptor) antibodies will be valuable

• Post-translational modifications:

  • Phosphorylation, acetylation, or other modifications of EHHADH may regulate its function

  • Development of modification-specific antibodies could reveal regulatory mechanisms

  • These would complement standard EHHADH antibodies in signaling studies

• EHHADH in docosahexaenoic acid (DHA) synthesis:

  • Recently discovered role of EHHADH in the "Sprecher" pathway for DHA synthesis

  • Antibodies that can detect specific EHHADH conformations during this process would advance understanding

  • Combined with metabolic labeling, these could reveal spatiotemporal dynamics of DHA synthesis

• Subcellular mislocalization in disease states:

  • In certain conditions, EHHADH may mislocalize from peroxisomes

  • High-resolution imaging with specialized antibodies could track this mislocalization

  • Particularly relevant in peroxisomal disorders and potentially in metabolic diseases

• EHHADH at the peroxisome-mitochondria interface:

  • Emerging evidence suggests crosstalk between these organelles

  • Proximity labeling combined with EHHADH antibodies could identify novel interaction partners

  • Super-resolution microscopy with appropriate antibodies could visualize these interactions

These emerging research areas will drive development of more specialized EHHADH antibody applications and techniques.

How might advances in antibody technology enhance EHHADH research in the coming years?

Emerging antibody technologies promise to revolutionize EHHADH research through several innovative approaches:

• Single-domain antibodies and nanobodies:

  • Smaller size allows better penetration of peroxisomal membranes

  • Potential for live-cell imaging of EHHADH dynamics

  • May enable super-resolution microscopy approaches with improved spatial resolution

• Recombinant antibody fragments:

  • Higher consistency and reproducibility compared to traditional polyclonal antibodies

  • Engineered specificity for particular EHHADH domains or conformational states

  • Potential for developing antibodies targeting specific EHHADH isoforms

• Intrabodies for in vivo tracking:

  • Expression of anti-EHHADH antibody fragments within living cells

  • Real-time monitoring of EHHADH localization and trafficking

  • Could reveal dynamic changes during peroxisomal proliferation or pexophagy

• Proximity labeling antibodies:

  • Anti-EHHADH antibodies conjugated to enzymes like BioID or APEX2

  • Would enable identification of transient interaction partners

  • Particularly valuable for mapping the complete EHHADH interactome in different metabolic states

• Multiplex imaging technologies:

  • Simultaneous detection of EHHADH with multiple peroxisomal proteins

  • Mass cytometry or multiplexed ion beam imaging with metal-conjugated antibodies

  • Would provide comprehensive spatial mapping of peroxisomal protein networks

These technological advances will significantly enhance the resolution, specificity, and functional insights that can be gained through EHHADH antibody applications in research.