ECI1 Antibody

What is ECI1 and what is its biological function?

ECI1, also known as enoyl-CoA delta isomerase 1 or DCI, is a key enzyme in fatty acid β-oxidation that catalyzes the conversion of cis- or trans-double bonds at the gamma carbon (position 3) to trans-double bonds at the beta carbon (position 2). This isomerization is particularly important for the metabolism of unsaturated fatty acids. ECI1 belongs to the hydratase/isomerase or crotonase superfamily of enzymes . It plays a critical role in maintaining cellular lipid homeostasis and is localized primarily in the mitochondrial matrix. The protein has a calculated molecular weight of approximately 33 kDa and contains a characteristic N-terminal core with a spiral fold composed of four turns, each consisting of two beta-sheets and one alpha-helix .

What types of ECI1 antibodies are available for research?

Several ECI1 antibodies are available for research applications, with polyclonal antibodies being most common. Examples include:

• ECI1 Rabbit Polyclonal Antibody (CAB1211) - Reactive against human and mouse ECI1, generated using a recombinant fusion protein containing amino acids 63-302 of human ECI1 .

• ECI1 Antibody (OABB00715) - A rabbit IgG polyclonal antibody reactive against human, mouse, and rat ECI1, produced using a polypeptide immunogen .

These antibodies are validated for various applications, including Western blot (WB), immunohistochemistry with paraffin-embedded sections (IHC-P), immunocytochemistry (ICC), and immunofluorescence (IF) .

How should I validate an ECI1 antibody before use in my experiments?

Antibody validation is critical for ensuring experimental reliability. For ECI1 antibodies, a multi-step validation approach is recommended:

• Specificity testing: Perform Western blots using positive control samples (e.g., HeLa or RAW 264.7 cell lysates) alongside negative controls. Verify that the antibody detects a band at the expected molecular weight (~33 kDa) .

• Knockdown/knockout validation: Compare antibody signals between wild-type samples and those with ECI1 knocked down or knocked out to confirm specificity.

• Cross-reactivity assessment: If working with non-human models, verify cross-reactivity with your species of interest. Available ECI1 antibodies show reactivity with human, mouse, and rat samples .

• Application-specific validation: For each intended application (WB, IHC-P, IF, etc.), optimize conditions using positive control samples and appropriate negative controls.

• Reproducibility testing: Perform replicate experiments to ensure consistent results across different batches and experimental conditions.

What are the optimal conditions for Western blotting with ECI1 antibodies?

For optimal Western blot results with ECI1 antibodies:

• Sample preparation: Extract proteins from tissues or cells using a lysis buffer containing protease inhibitors. For mitochondrial proteins like ECI1, consider using a mitochondrial isolation protocol for enrichment.

• Protein loading: Load 20-50 μg of total protein per lane.

• Gel electrophoresis: Use 10-12% SDS-PAGE gels for optimal separation around the 33 kDa range.

• Transfer conditions: Transfer to PVDF or nitrocellulose membranes at 100V for 60-90 minutes in standard transfer buffer.

• Blocking: Block membranes in 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody: Dilute ECI1 antibodies to the recommended concentration (typically 1:500-1:2000 for WB applications) . Incubate overnight at 4°C.

• Washing: Wash membranes 3-5 times with TBST, 5-10 minutes each.

• Secondary antibody: Use appropriate HRP-conjugated secondary antibody (anti-rabbit for the polyclonal antibodies mentioned) at 1:5000-1:10000 dilution.

• Detection: Visualize using ECL detection reagents and document using an imaging system capable of detecting chemiluminescence.

What are the recommended protocols for immunohistochemistry with ECI1 antibodies?

For immunohistochemistry with paraffin-embedded tissue sections:

• Tissue preparation: Fix tissues in 10% neutral buffered formalin and embed in paraffin. Cut 4-6 μm sections and mount on positively charged slides.

• Deparaffinization: Treat slides with xylene and rehydrate through graded alcohols to water.

• Antigen retrieval: This is crucial for ECI1 detection. Heat slides in 10 mM citrate buffer (pH 6.0) for 20 minutes . Allow to cool slowly to room temperature.

• Endogenous peroxidase blocking: Treat with 3% hydrogen peroxide for 10 minutes.

• Protein blocking: Block with serum-free protein block for 10-20 minutes.

• Primary antibody: Apply diluted ECI1 antibody (1:50-1:200 recommended for IHC-P) and incubate overnight at 4°C or for 1 hour at room temperature.

• Detection system: Use a polymer-based detection system appropriate for rabbit primary antibodies.

• Counterstaining: Counterstain with hematoxylin, dehydrate, clear, and mount.

• Controls: Include positive controls (prostate cancer tissue shows strong ECI1 expression ) and negative controls (primary antibody omitted).

How can I use ECI1 antibodies to study its role in cancer progression?

ECI1 has emerged as a potential biomarker and therapeutic target in prostate cancer . To investigate its role in cancer progression:

What approaches can I use to study ECI1's role in fatty acid metabolism?

To investigate ECI1's function in fatty acid metabolism:

• Metabolic flux analysis: Use stable isotope-labeled fatty acids (e.g., 13C-labeled) to trace metabolic pathways in cells with normal, elevated, or reduced ECI1 expression. Analyze by mass spectrometry to determine how ECI1 levels affect fatty acid oxidation rates and metabolite production.

• Mitochondrial function assessment: Measure oxygen consumption rates using respirometry (e.g., Seahorse XF Analyzer) in cells with varying ECI1 expression. Research shows ECI1 enhances maximal mitochondrial respiratory capacity .

• Lipid profiling: Perform lipidomic analysis to identify changes in lipid species and fatty acid composition associated with ECI1 expression levels.

• Subcellular localization studies: Use immunofluorescence with ECI1 antibodies (recommended dilution 1:50-1:200) combined with mitochondrial markers to study its localization within the mitochondrial matrix and potential redistribution under different metabolic conditions.

• Protein-protein interaction studies: Employ immunoprecipitation with ECI1 antibodies followed by mass spectrometry to identify interacting partners in the fatty acid β-oxidation pathway and other potential metabolic networks.

How can I correlate ECI1 expression with genomic alterations at the 16p13.3 locus?

To investigate the relationship between 16p13.3 genomic alterations and ECI1 expression:

• Genomic analysis: Perform chromosomal microarray analysis (CMA) or next-generation sequencing to identify copy number variations at the 16p13.3 locus in patient samples or cell lines.

• Expression correlation: Use quantitative PCR, Western blotting with ECI1 antibodies, or IHC to measure ECI1 expression levels in samples with and without 16p13.3 gain. Previous research has demonstrated higher ECI1 expression in prostate cancer cases with 16p13.3 gain .

• Functional validation: Create cell models with artificially induced 16p13.3 gain using CRISPR-based techniques, then assess changes in ECI1 expression and cellular phenotypes.

• Multi-omics integration: Combine genomic data with transcriptomic, proteomic, and metabolomic analyses to understand how 16p13.3 gains affect not only ECI1 expression but also broader cellular pathways.

• Clinical correlation: Analyze patient cohorts to determine if 16p13.3 gain and ECI1 overexpression together provide stronger prognostic information than either marker alone.

Why might I see multiple bands when using ECI1 antibodies in Western blot?

Multiple bands in Western blots using ECI1 antibodies could result from:

• Post-translational modifications: ECI1 may undergo modifications such as phosphorylation or ubiquitination, resulting in higher molecular weight bands.

• Protein isoforms: Alternative splicing could generate multiple ECI1 isoforms of different sizes.

• Protein degradation: Proteolytic cleavage during sample preparation may generate lower molecular weight fragments. Ensure complete protease inhibition during sample preparation.

• Non-specific binding: The antibody may cross-react with structurally similar proteins, particularly other members of the hydratase/isomerase superfamily. Try more stringent washing conditions or higher antibody dilutions.

• Incomplete denaturation: Ensure complete sample denaturation by boiling in sample buffer containing sufficient SDS and reducing agent.

To address these issues:

• Compare bands with the expected molecular weight (33 kDa for ECI1)

• Include appropriate positive and negative controls

• Consider antibody validation using ECI1 knockout or knockdown samples

• Optimize antibody dilution, typically starting with 1:500-1:2000 for Western blotting

How should I interpret heterogeneous ECI1 staining patterns in immunohistochemistry?

Heterogeneous ECI1 staining patterns in IHC may provide important biological insights:

• Subcellular localization: ECI1 is primarily localized to the mitochondrial matrix . Confirm that staining shows the expected punctate cytoplasmic pattern consistent with mitochondrial localization.

• Tissue heterogeneity: Variations in staining intensity within a tissue may reflect biological heterogeneity. In prostate cancer, for example, ECI1 expression varies among tumors and is higher in cases with 16p13.3 gain, high Gleason grade, and advanced tumor stage .

• Stromal vs. epithelial staining: Evaluate whether ECI1 staining differs between stromal and epithelial components. This may provide insights into tissue-specific metabolic differences.

• Technical considerations: Heterogeneous staining might result from fixation artifacts, antigen retrieval variations, or antibody penetration issues. Ensure consistent tissue processing and staining protocols.

For reliable interpretation:

• Use a scoring system that accounts for both staining intensity and percentage of positive cells

• Compare with normal adjacent tissue controls

• Correlate with other metabolic markers

• Consider digital pathology tools for quantitative analysis of staining patterns

ECI1 Antibody Application Reference Table

Data compiled from antibody specifications for ECI1 Rabbit Polyclonal Antibody (CAB1211) and ECI1 Antibody (OABB00715) .

ECI1 Expression in Prostate Cancer Progression

Data summarized from research findings on ECI1's role in prostate cancer progression .