ECI2 Antibody, HRP conjugated

What is ECI2 and why is it studied in research applications?

ECI2 (Enoyl-CoA Delta Isomerase 2) is a mitochondrial enzyme that plays a crucial role in fatty acid metabolism. This protein is capable of isomerizing both 3-cis and 3-trans double bonds into the 2-trans form across various enoyl-CoA species, with a notable preference for 3-trans substrates . ECI2 is also known by several synonyms including DRS-1, pECI, and hepatocellular carcinoma-associated antigen 88 . The enzyme's involvement in lipid metabolism pathways makes it a significant target for research in metabolic disorders, certain cancers, and mitochondrial function studies. The UniProt accession number for human ECI2 is O75521 , providing researchers with a standardized reference point for additional protein information.

What applications is the ECI2 Antibody, HRP conjugated typically used for?

ECI2 Antibody, HRP conjugated is primarily optimized for ELISA applications according to manufacturer specifications . While ELISA represents the validated application, researchers should note that HRP-conjugated antibodies are generally suitable for multiple detection methods including chemiluminescent, colorimetric, and fluorescent detection systems . The direct conjugation of HRP to the primary antibody eliminates the need for secondary antibody incubation, which can be advantageous in time-sensitive experimental designs or when reducing protocol complexity is desired. Before applying this antibody to non-validated applications such as western blotting or immunohistochemistry, preliminary optimization experiments should be conducted to determine appropriate working dilutions and conditions.

What are the key advantages and limitations of using HRP-conjugated primary antibodies compared to conventional two-step detection systems?

When deciding between HRP-conjugated primary antibodies and conventional primary-secondary systems, researchers should consider several methodological trade-offs:

What optimization steps should be taken when using ECI2 Antibody, HRP conjugated in ELISA assays?

Optimizing ELISA protocols with ECI2 Antibody, HRP conjugated requires systematic adjustment of several parameters:

• Antibody titration: Though manufacturer-recommended dilutions provide starting points, performing a titration series (typically 1:500, 1:1000, 1:2000, 1:5000, and 1:10000) is essential to determine the optimal antibody concentration that maximizes specific signal while minimizing background.

• Blocking optimization: Test different blocking reagents (BSA, milk proteins, commercial blockers) at various concentrations (1-5%) to identify the most effective combination for reducing non-specific binding.

• Incubation conditions: Experiment with various incubation times (1-4 hours) and temperatures (room temperature vs. 4°C) for the antibody binding step to enhance sensitivity.

• Substrate selection: For HRP detection, compare chemiluminescent substrates (for highest sensitivity), chromogenic substrates (for visual quantification), or fluorogenic substrates based on experimental requirements .

• Washing optimization: Determine optimal washing buffer composition (PBS-T or TBS-T) and washing frequency to remove unbound antibody without disrupting specific interactions.

Documenting each optimization step in a structured experimental design allows for systematic improvement of assay performance and reproducibility across experiments.

How can the specificity of ECI2 Antibody, HRP conjugated be validated in experimental systems?

Validating antibody specificity is critical for ensuring reliable research outcomes. For ECI2 Antibody, HRP conjugated, consider implementing these validation strategies:

• Positive and negative controls: Include samples with known ECI2 expression levels alongside samples where ECI2 is absent or knocked down.

• Epitope competition assay: Pre-incubate the antibody with excess immunogen peptide (recombinant Human Enoyl-CoA delta isomerase 2, mitochondrial protein, amino acids 119-219) before application to samples. Specific binding should be blocked by the peptide competition.

• Cross-reactivity assessment: Test the antibody against closely related proteins (particularly other enoyl-CoA isomerases) to confirm specificity within the protein family.

• Knockout/knockdown validation: Compare signals between wild-type samples and those where ECI2 has been genetically knocked out or knocked down via RNA interference.

• Molecular weight verification: When using in western blot applications (if validated), confirm that the detected band corresponds to the expected molecular weight of ECI2.

These validation steps should be performed for each new lot of antibody and for each experimental system to ensure consistent performance across studies.

What troubleshooting approaches are recommended when encountering weak signals with ECI2 Antibody, HRP conjugated?

When weak signals are observed using ECI2 Antibody, HRP conjugated, consider these advanced troubleshooting strategies:

• Signal enhancement options: If target protein levels are low, consider switching to the indirect detection method (primary + secondary antibody approach) for signal amplification, as this can provide significantly higher sensitivity compared to direct conjugated antibodies .

• Substrate optimization: Experiment with more sensitive HRP substrates. Enhanced chemiluminescent substrates often provide significantly higher sensitivity than standard formulations .

• Protein enrichment: For applications where ECI2 concentration may be low, implement subcellular fractionation to isolate mitochondrial fractions where ECI2 is predominantly localized .

• Increased antibody concentration: While maintaining specificity, gradually increase antibody concentration to improve signal. Monitor background levels to ensure signal-to-noise ratio remains optimal.

• Sample handling improvements: Ensure proper sample preparation with appropriate protease inhibitors to prevent degradation of the target protein during extraction and processing.

• Extended exposure times: For chemiluminescent detection, test multiple exposure times to capture optimal signal development without saturation.

Documentation of all troubleshooting steps within a systematic experimental framework allows for reproducible optimization across different experimental conditions.

What storage and handling practices maximize the shelf-life and performance of ECI2 Antibody, HRP conjugated?

Proper storage and handling of HRP-conjugated antibodies is critical for maintaining long-term performance. For ECI2 Antibody, HRP conjugated, implement these evidence-based practices:

• Storage temperature: Store at -20°C as recommended by manufacturers . Avoid repeated freeze-thaw cycles as they can degrade both the antibody and the conjugated HRP enzyme.

• Aliquoting strategy: Upon receipt, divide the antibody into small working aliquots (10-20 μL) to minimize freeze-thaw cycles and prevent contamination of the stock.

• Light sensitivity: Protect HRP-conjugated antibodies from prolonged light exposure as this can degrade the enzyme activity and reduce signal strength .

• Buffer considerations: The antibody is typically supplied in a stabilizing buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . Avoid introducing contaminants that may compromise this protective environment.

• Working dilution preparation: Prepare fresh working dilutions on the day of experiment rather than storing diluted antibody for extended periods, as protein adsorption to tube walls and HRP degradation can occur in dilute solutions.

• Functional validation: Periodically validate the activity of stored antibody against a reference sample to ensure consistent performance over time.

Following these storage and handling recommendations will help maintain optimal antibody performance throughout its shelf-life and experimental usage.

What considerations are important when using ECI2 Antibody, HRP conjugated in multiplex detection systems?

When incorporating ECI2 Antibody, HRP conjugated into multiplex detection systems, researchers should address several technical challenges:

• Cross-reactivity assessment: Thoroughly test for potential cross-reactivity with other primary antibodies in the multiplex panel, particularly those raised in the same host species (rabbit) .

• Signal separation strategies: Since HRP produces a single type of signal, true multiplexing with spectral separation requires combining with antibodies conjugated to different enzymes or fluorophores. Consider sequential detection methods if using multiple HRP-conjugated antibodies.

• Enzyme inactivation protocols: When performing sequential HRP detections, implement complete HRP inactivation between rounds using hydrogen peroxide solutions (3-10%) or other validated inactivation methods.

• Spatial separation techniques: For tissue-based applications, consider using antibodies against proteins with distinct subcellular localizations to achieve spatial separation of signals even with the same detection system.

• Optimization of concentration ratios: When multiple antibodies are used simultaneously, the relative concentrations may need adjustment from single-plex conditions to account for competitive binding or background effects.

These considerations ensure that multiplex detection systems maintain specificity and sensitivity when incorporating ECI2 Antibody, HRP conjugated alongside other detection reagents.

How can ECI2 Antibody, HRP conjugated be integrated into studies examining fatty acid metabolism and related diseases?

The integration of ECI2 Antibody, HRP conjugated into fatty acid metabolism research requires thoughtful experimental design:

• Pathophysiological models: Design experiments comparing ECI2 expression across healthy controls and disease models related to fatty acid metabolism disorders, particularly those affecting mitochondrial β-oxidation pathways.

• Intervention studies: Measure changes in ECI2 levels before and after pharmaceutical interventions targeting fatty acid metabolism to establish correlations between treatment efficacy and enzyme expression.

• Co-expression analysis: Implement co-immunoprecipitation studies or proximity ligation assays to investigate protein-protein interactions between ECI2 and other components of the fatty acid metabolic machinery.

• Subcellular localization mapping: Use the antibody in conjunction with organelle markers to track potential changes in ECI2 distribution between mitochondria and peroxisomes under different metabolic conditions.

• Quantitative analysis pipelines: Develop standardized quantification protocols for ELISA data to enable reliable comparison of ECI2 levels across experimental conditions and between research groups.

This systematic approach allows researchers to generate meaningful insights into the role of ECI2 in normal physiology and pathological states related to lipid metabolism.

What are the most effective strategies for reducing background and non-specific binding when using ECI2 Antibody, HRP conjugated?

Achieving optimal signal-to-noise ratios with ECI2 Antibody, HRP conjugated requires implementation of advanced background reduction strategies:

• Blocking optimization matrix: Systematically test combinations of blocking agents (BSA, casein, commercial blockers) at various concentrations (1-5%) and incubation times (30 minutes to overnight) to identify optimal conditions for your specific sample type.

• Diluent formulation refinement: Experiment with different antibody diluents containing varying salt concentrations (150-500 mM NaCl) and detergent levels (0.05-0.1% Tween-20) to reduce non-specific hydrophobic and ionic interactions.

• Pre-absorption techniques: For tissues with known cross-reactivity issues, pre-absorb the antibody with tissue homogenates from negative control samples to deplete cross-reactive antibodies before application to experimental samples.

• Endogenous enzyme blocking: When using tissue samples, implement specific blocking of endogenous peroxidase activity using hydrogen peroxide treatment (0.3-3% H₂O₂ for 10-30 minutes) before antibody application.

• Washing buffer optimization: Compare different washing buffer compositions (PBS vs. TBS) and detergent concentrations to identify conditions that effectively remove unbound antibody while preserving specific interactions.

• Signal thresholding methods: Implement computational approaches for quantitative applications to distinguish specific signal from background based on statistical thresholds derived from negative controls.

These strategies should be systematically tested and optimized for each specific application and sample type to achieve consistent, high-quality results.