ECI2 Antibody, Biotin conjugated

What is ECI2 and why is it a target for antibody research?

ECI2 (Enoyl-CoA Delta Isomerase 2), also known as ACBD2 (Acyl-CoA Binding Domain Containing 2), is a protein involved in fatty acid metabolism that plays a significant role in the isomerization of unsaturated fatty acids. It contains both an ECI/enoyl-CoA hydratase (ECH) domain and an ACBD domain, both relevant to fatty acid metabolism . ECI2 has become an important research target due to its dual localization in both peroxisomes and mitochondria, making it valuable for studying organelle interactions and metabolic pathways .

The protein is cataloged in multiple biological databases, including:

• HGNC: 14601

• OMIM: 608024

• KEGG: hsa:10455

• STRING: 9606.ENSP00000369461

• UniGene: Hs.15250

These database entries facilitate comprehensive investigation of ECI2's structural and functional characteristics across different research platforms.

How does biotin conjugation enhance the functionality of ECI2 antibody in research applications?

Biotin conjugation significantly enhances the utility of ECI2 antibody through several key mechanisms:

• The biotin-streptavidin interaction is one of the strongest non-covalent biological interactions known, with extremely high affinity, providing robust and specific detection .

• Biotin conjugation allows for signal amplification, as multiple streptavidin molecules (linked to detection reagents) can bind to each biotinylated antibody, enhancing sensitivity particularly in samples with low target expression .

• The small size of biotin ensures minimal interference with antibody binding to ECI2, preserving the antibody's specificity and affinity .

• Biotin-SP conjugates, which contain a 6-atom spacer between biotin and the antibody, further increase sensitivity by extending the biotin moiety away from the antibody surface, making it more accessible to streptavidin binding sites .

These properties make ECI2 Antibody, Biotin conjugated particularly valuable for detecting low-abundance ECI2 in complex cellular environments where it may be distributed between peroxisomes and mitochondria.

What are the recommended applications for ECI2 Antibody, Biotin conjugated?

ECI2 Antibody, Biotin conjugated is suitable for multiple experimental applications:

The antibody is especially valuable for investigating the subcellular distribution of ECI2/ACBD2 in studies examining peroxisome-mitochondria interactions . When designing experiments, researchers should consider ECI2's dual localization pattern and include appropriate subcellular markers for colocalization studies.

What detection systems work best with ECI2 Antibody, Biotin conjugated?

The biotinylated ECI2 antibody requires secondary detection reagents based on avidin/streptavidin affinity. Several detection systems offer distinct advantages:

• Streptavidin-HRP (horseradish peroxidase): Provides excellent sensitivity for chromogenic detection in immunohistochemistry and western blotting applications .

• Streptavidin-fluorophore conjugates: Enable direct fluorescent visualization in immunofluorescence studies, with options including streptavidin-FITC, streptavidin-Cy3, or streptavidin-Alexa Fluor dyes for multicolor applications .

• Streptavidin-alkaline phosphatase: Particularly beneficial when used with Biotin-SP conjugates, which employ a 6-atom spacer that increases accessibility of biotin to streptavidin binding sites, resulting in enhanced sensitivity .

• Amplification systems: For detecting low-abundance ECI2, tyramide signal amplification (TSA) systems compatible with biotinylated antibodies can significantly enhance signal detection while maintaining specificity.

When selecting a detection system, consider the cellular compartments being investigated, as peroxisomal and mitochondrial localization of ECI2 may require different sensitivity levels for optimal visualization.

How should ECI2 Antibody, Biotin conjugated be stored and handled to maintain optimal activity?

To maintain optimal activity of ECI2 Antibody, Biotin conjugated:

• Storage temperature: Store at -20°C for long-term stability (up to 12 months under proper conditions) .

• Storage buffer: The antibody is typically provided in an aqueous buffered solution containing 0.01M TBS (pH 7.4) with 1% BSA, 0.02% Proclin300, and 50% Glycerol, which helps maintain antibody stability and prevent microbial growth .

• Freeze-thaw cycles: Minimize repeated freeze-thaw cycles by aliquoting the antibody into smaller volumes before freezing.

• Working dilutions: Prepare fresh working dilutions on the day of the experiment, as diluted antibody solutions may lose activity over time.

• Light sensitivity: While the biotin conjugate itself is not light-sensitive, any streptavidin-fluorophore detection reagents should be protected from light to prevent photobleaching.

• Temperature during assays: Maintain consistent temperature during incubation steps; most protocols recommend room temperature or 4°C depending on the specific application.

Following these guidelines will help ensure reproducible results across experiments and maximize the shelf-life of the antibody.

How can ECI2 Antibody, Biotin conjugated be utilized to study peroxisome-mitochondria interactions?

ECI2 Antibody, Biotin conjugated serves as a valuable tool for investigating peroxisome-mitochondria interactions due to ECI2's unique dual localization pattern. Research strategies include:

• Co-immunoprecipitation studies: ECI2 Antibody, Biotin conjugated can be used to identify protein-protein interactions at the peroxisome-mitochondria interface. Research has demonstrated that in-cell co-IP methodology can validate the proximity between peroxisomal ECI2/ACBD2 and mitochondrial import receptors like TOMM20 .

• Dual labeling approaches: Combining ECI2 Antibody, Biotin conjugated with markers for peroxisomes (e.g., anti-SCP2) and mitochondria (e.g., anti-COX IV) enables visualization of "peroxisome-like structures" that mediate organelle contact . This requires:

  • Streptavidin-conjugated fluorophore for ECI2 detection

  • Differentially labeled antibodies for peroxisomal and mitochondrial markers

  • High-resolution confocal or super-resolution microscopy

• Functional analysis: ECI2 Antibody, Biotin conjugated can track changes in ECI2 localization during metabolic stress or hormone stimulation, which has been shown to affect steroid biosynthesis. Ectopic expression of ACBD2/ECI2 isoform A led to increased basal and hormone-stimulated steroid formation, suggesting that ECI2-mediated peroxisome-mitochondria interactions facilitate metabolite exchange between these organelles .

• Proximity ligation assays: When combined with antibodies against peroxisomal import receptor PEX5 or mitochondrial import receptor TOMM20, biotinylated ECI2 antibody can help quantify changes in organelle proximity under different experimental conditions.

These approaches have revealed that peroxisome-mitochondria contact occurs via specialized structures involving peroxisomal and mitochondrial matrix protein import complexes, with ECI2/ACBD2 playing a mediating role in this interaction .

What methodological considerations are important when using ECI2 Antibody, Biotin conjugated for examining fatty acid metabolism?

When investigating fatty acid metabolism with ECI2 Antibody, Biotin conjugated, several methodological considerations are essential:

• Functional redundancy assessment: ECI2 has similar functions to ECI1 in unsaturated fatty acid metabolism. When designing experiments, consider that Eci1 knockout mice show no pronounced phenotype, but knockdown of Eci2 in Eci1-deficient fibroblasts results in C12:1 acylcarnitine accumulation . This functional overlap necessitates careful experimental design to distinguish ECI2-specific effects.

• Isoform-specific analysis: Multiple ECI2 isoforms exist with potentially different localizations and functions. The ECI2 isoform A has been specifically linked to increased steroid formation when ectopically expressed . Ensure your experimental design accounts for potential isoform-specific effects.

• Metabolic state considerations: The subcellular distribution of ECI2 may vary depending on the cell's metabolic state. Design time-course experiments to capture dynamic changes in ECI2 localization during different metabolic conditions.

• Quantification approaches:

  • For western blotting, include mitochondrial (COX IV) and peroxisomal (SCP2) markers for fraction purity assessment

  • Use GAPDH as a whole protein loading control

  • Consider densitometric analysis to quantify relative distribution between compartments

• Cell/tissue type selection: The role of ECI2 in steroid biosynthesis makes steroidogenic cells (like Leydig cells) particularly relevant models. Studies have used both MA-10 mouse tumor Leydig cells and mLTC-1 mouse tumor Leydig cells successfully .

These methodological considerations will help ensure robust and reproducible results when investigating ECI2's role in fatty acid metabolism.

How can signal amplification techniques enhance detection sensitivity when using ECI2 Antibody, Biotin conjugated?

Signal amplification techniques can significantly enhance detection sensitivity for ECI2 Antibody, Biotin conjugated, particularly when investigating low-abundance targets or subtle changes in subcellular distribution:

• Avidin-Biotin Complex (ABC) method:

  • After primary incubation with ECI2 Antibody, Biotin conjugated, apply a preformed complex of avidin-biotinylated enzyme

  • Multiple enzyme molecules per complex increase signal output

  • Particularly effective for chromogenic detection in immunohistochemistry applications

• Tyramide Signal Amplification (TSA):

  • Uses the catalytic activity of HRP to generate high-density labeling

  • Process: Biotinylated ECI2 antibody → Streptavidin-HRP → Tyramide substrate activation → Covalent binding of labeled tyramide to proteins near HRP

  • Can increase sensitivity 10-100 fold compared to conventional detection methods

  • Especially valuable for detecting low abundance of ECI2 in specific subcellular compartments

• Biotin-SP conjugated systems:

  • The 6-atom spacer in Biotin-SP extends the biotin moiety away from the antibody surface

  • This increased accessibility to streptavidin binding sites enhances sensitivity

  • Particularly notable when used with alkaline phosphatase-conjugated streptavidin

• Sequential multiple labeling:

  • Apply and elute/photobleach detection reagents sequentially

  • Allows visualization of multiple targets using the same fluorophore

  • Useful for colocalization studies of ECI2 with other peroxisomal or mitochondrial proteins

When implementing these techniques, optimization of incubation times, reagent concentrations, and washing steps is essential to maximize signal-to-noise ratio while maintaining specificity.

What experimental approaches can validate the specificity of ECI2 Antibody, Biotin conjugated in complex cellular environments?

• Genetic controls:

  • siRNA or shRNA knockdown of ECI2/ACBD2 should reduce or eliminate signal

  • CRISPR-Cas9 knockout cells provide definitive negative controls

  • Overexpression systems with tagged ECI2 can confirm antibody recognition of the target

• Peptide competition assays:

  • Pre-incubate the antibody with increasing concentrations of the immunizing peptide

  • Specific binding should be competitively inhibited in a dose-dependent manner

  • Include non-specific peptides as negative controls

• Cross-validation with multiple antibodies:

  • Compare staining patterns with non-biotinylated ECI2 antibodies

  • Use antibodies targeting different epitopes of ECI2

  • Confirm subcellular localization with established organelle markers (peroxisomal SCP2, mitochondrial COX IV)

• Western blot analysis:

  • Confirm single band of expected molecular weight (approximately 58 kDa for ECI2)

  • Include subcellular fractionation to confirm dual localization in peroxisomes and mitochondria

  • Compare with recombinant ECI2 protein as a positive control

• Mass spectrometry validation:

  • Perform immunoprecipitation using ECI2 Antibody, Biotin conjugated

  • Analyze pulled-down proteins by mass spectrometry

  • Confirm presence of ECI2/ACBD2 peptides in the immunoprecipitates

These validation approaches should be documented thoroughly and included in research publications to strengthen the reliability of findings based on this antibody.

How can ECI2 Antibody, Biotin conjugated contribute to understanding the role of ECI2 in steroid biosynthesis?

ECI2 Antibody, Biotin conjugated offers powerful methodological approaches for investigating ECI2's role in steroid biosynthesis:

• Spatiotemporal analysis of ECI2 during steroidogenesis:

  • Track changes in ECI2 localization during hormone stimulation using time-course immunofluorescence

  • Quantify redistribution between peroxisomes and mitochondria

  • Research has shown that ectopic expression of ACBD2/ECI2 isoform A in MA-10 cells increases both basal and hormone-stimulated steroid formation

• Organelle interaction studies:

  • Visualize "peroxisome-like structures" that mediate contact between organelles

  • Use dual-labeling approaches combining ECI2 Antibody, Biotin conjugated with markers for steroidogenic enzymes

  • The close proximity between peroxisomal ECI2/ACBD2 and mitochondrial TOMM20 suggests a mechanism for metabolite exchange that supports steroid biosynthesis

• Co-immunoprecipitation analysis:

  • Identify protein complexes involving ECI2 during different stages of steroidogenesis

  • In-cell co-IP methodology has successfully validated interactions between ECI2 and organelle import receptors

  • This approach can uncover additional binding partners that may regulate ECI2's role in steroid biosynthesis

• Functional correlation studies:

  • Combine immunolocalization of ECI2 with measurements of steroid hormone production

  • Assess how manipulations of ECI2 expression affect steroidogenic enzyme activity

  • Investigate whether ECI2-mediated peroxisome-mitochondria contacts facilitate the exchange of specific metabolites needed for steroid biosynthesis

• Tissue-specific expression analysis:

  • Compare ECI2 expression and localization across different steroidogenic tissues

  • Mouse tumor Leydig cells (MA-10 and mLTC-1) have been successfully used as model systems

  • Correlation between ECI2 expression levels and steroidogenic capacity can provide insights into its functional significance

These approaches collectively address how ECI2-mediated peroxisome-mitochondria interactions favor the exchange of metabolites and/or macromolecules between these organelles in support of steroid biosynthesis .

How do I resolve high background issues when using ECI2 Antibody, Biotin conjugated in immunohistochemistry?

High background is a common challenge when using biotinylated antibodies. For ECI2 Antibody, Biotin conjugated, consider these troubleshooting approaches:

• Block endogenous biotin:

  • Tissue samples, especially liver, kidney, and many tumor tissues, contain high levels of endogenous biotin

  • Pre-block tissues with avidin followed by biotin before applying biotinylated antibodies

  • Commercial avidin/biotin blocking kits are available and should be used according to manufacturer protocols

• Optimize blocking conditions:

  • Increase blocking solution concentration (try 5-10% normal serum from the same species as the secondary reagent)

  • Extend blocking time to 1-2 hours at room temperature

  • Add 0.1-0.3% Triton X-100 to blocking solution for improved penetration

• Titrate antibody concentration:

  • Test multiple dilutions within the recommended range (1:20-1:200 for IHC)

  • Always prepare fresh working dilutions on the day of the experiment

  • Consider adjusting incubation time and temperature (overnight at 4°C may reduce non-specific binding)

• Improve washing protocols:

  • Increase number of washes (5-6 washes of 5-10 minutes each)

  • Use PBS-T (PBS with 0.05-0.1% Tween-20) for more effective washing

  • Ensure thorough washing between all steps, especially after the biotinylated antibody

• Modify detection system:

  • If using ABC systems, dilute the avidin-biotin-enzyme complex further

  • For fluorescent detection, try directly labeled streptavidin conjugates instead of amplification systems

  • Consider non-biotin detection systems if endogenous biotin persists as a problem

These approaches should be tested systematically, changing one variable at a time to determine the optimal conditions for your specific tissue and experimental setup.

What controls should be included when designing experiments with ECI2 Antibody, Biotin conjugated?

A comprehensive set of controls is essential for rigorous experimental design with ECI2 Antibody, Biotin conjugated:

• Primary antibody controls:

  • Negative control: Omit primary antibody but include all other reagents

  • Isotype control: Use non-specific IgG of the same isotype and concentration as ECI2 antibody

  • Peptide competition: Pre-incubate antibody with immunizing peptide to demonstrate specificity

• Sample-specific controls:

  • Positive tissue/cell control: Include samples known to express ECI2 (e.g., steroidogenic cells)

  • Negative tissue/cell control: Include samples with minimal ECI2 expression

  • Genetic controls: Where possible, include ECI2 knockdown or knockout samples

• Biotin-specific controls:

  • Endogenous biotin control: Process a sample with only streptavidin-detection reagent (no antibody)

  • Avidin/biotin blocking control: Compare samples with and without avidin/biotin blocking

  • Non-biotinylated antibody control: Compare signals between biotinylated and non-biotinylated versions of the same antibody

• Subcellular localization controls:

  • Co-staining with peroxisomal markers (e.g., SCP2)

  • Co-staining with mitochondrial markers (e.g., COX IV)

  • Include GAPDH staining as a cytosolic reference

• Method-specific controls:

  • For Western blotting: Include recombinant ECI2 protein or cell lysates with known ECI2 expression

  • For immunoprecipitation: Include a "beads only" control without antibody

  • For ELISA: Include standard curves and blank wells

What optimization strategies improve results when using ECI2 Antibody, Biotin conjugated with streptavidin-based detection systems?

Optimizing streptavidin-based detection with ECI2 Antibody, Biotin conjugated requires attention to several key parameters:

• Streptavidin conjugate selection:

  • For highest sensitivity in chromogenic detection: HRP-conjugated streptavidin with tyramide amplification

  • For multicolor fluorescence: Choose fluorophores with minimal spectral overlap with other channels

  • For applications requiring increased sensitivity: Biotin-SP (with 6-atom spacer) shows enhanced performance with alkaline phosphatase-conjugated streptavidin

• Incubation parameters:

  • Streptavidin concentration: Titrate to determine optimal concentration (typically 1-5 μg/mL)

  • Incubation time: Usually 30-60 minutes is sufficient; longer incubations may increase background

  • Temperature: Room temperature is standard, but 4°C may reduce non-specific binding

• Buffer optimization:

  • Include 0.1% BSA in streptavidin dilution buffer to reduce non-specific binding

  • Add 0.05-0.1% Tween-20 to wash buffers to remove unbound reagents effectively

  • Consider adding 0.3M NaCl to streptavidin dilution buffer to reduce ionic interactions

• Sequential application strategies:

  • For dual or triple labeling: Apply and detect primary antibodies sequentially rather than simultaneously

  • When using multiple biotinylated primary antibodies: Consider complete blocking or stripping between rounds

  • Alternative approach: Use ECI2 Antibody, Biotin conjugated with one detection system and directly labeled antibodies for other targets

• Amplification system optimization:

  • For ABC method: Prepare complex 30 minutes before use for optimal formation

  • For TSA systems: Carefully titrate tyramide reagent and limit reaction time to prevent excessive signal

  • For enzyme-based detection: Optimize substrate development time with frequent monitoring

These optimization strategies should be adapted to your specific experimental context, particularly considering ECI2's dual localization in peroxisomes and mitochondria, which may require different sensitivity levels for complete visualization .

How should sample preparation be modified for optimal detection of ECI2 in different cellular compartments?

ECI2's dual localization in peroxisomes and mitochondria necessitates specific sample preparation approaches for comprehensive detection:

• Fixation optimization:

  • For peroxisomal ECI2: 4% paraformaldehyde (10-15 minutes) preserves structure while maintaining antigenicity

  • For mitochondrial ECI2: Brief fixation (5-10 minutes) with lower paraformaldehyde concentration (2-3%) may improve epitope accessibility

  • Avoid methanol fixation which can extract lipids and disrupt membrane-associated proteins

• Permeabilization strategies:

  • Gentle detergents (0.1-0.3% Triton X-100) for balanced permeabilization of both organelles

  • Digitonin (25-50 μg/mL) for selective plasma membrane permeabilization to preserve organelle integrity

  • Saponin (0.1-0.2%) for reversible permeabilization that maintains organelle structural integrity

• Antigen retrieval methods:

  • Heat-induced epitope retrieval: Citrate buffer (pH 6.0) at 95-100°C for 15-20 minutes

  • Enzymatic retrieval: Proteinase K (10-20 μg/mL) for 5-10 minutes at room temperature

  • Test multiple methods, as ECI2 epitopes in different compartments may respond differently

• Subcellular fractionation:

  • For biochemical analysis, prepare crude mitochondrial fractions following established protocols

  • Verify fraction purity using compartment-specific markers: COX IV (mitochondria) and SCP2 (peroxisomes)

  • Use GAPDH as a cytosolic contamination marker

• Tissue-specific considerations:

  • Steroidogenic tissues (testes, adrenals): Minimize processing time to preserve metabolic state

  • Liver samples: Include avidin-biotin blocking steps to manage high endogenous biotin

  • Cultured cells: Consider live-cell imaging with membrane-permeable streptavidin conjugates for dynamic studies

Research has shown that ECI2/ACBD2 mediates peroxisome-mitochondria contact via specialized peroxisome-like structures . Optimizing sample preparation to preserve these delicate structural arrangements is essential for comprehensive analysis of ECI2's distribution and function.

What are the most effective strategies for multiplexing when using ECI2 Antibody, Biotin conjugated alongside other antibodies?

Effective multiplexing with ECI2 Antibody, Biotin conjugated requires careful planning to avoid cross-reactivity and signal interference:

• Sequential staining protocols:

  • Apply, detect, and block each primary antibody sequentially

  • For fluorescence applications: Consider photobleaching between rounds to eliminate signal overlap

  • For chromogenic detection: Use different enzyme systems (HRP, AP) with distinct substrates

• Species selection strategies:

  • Choose primary antibodies raised in different host species

  • Example combinations working with ECI2 Antibody, Biotin conjugated (raised in rabbit):

    • Mouse monoclonal antibodies against mitochondrial markers (e.g., anti-TOMM20)

    • Goat polyclonal antibodies against peroxisomal markers

• Direct labeling approaches:

  • Use directly labeled antibodies for some targets to reduce detection complexity

  • Consider zenon labeling technology for direct labeling of primary antibodies

  • Combine with biotinylated ECI2 antibody for enhanced flexibility

• Tyramide-based multiplexing:

  • Apply sequential tyramide signal amplification for each target

  • Between rounds, inactivate HRP (using hydrogen peroxide) while preserving deposited tyramide

  • This approach allows use of multiple primary antibodies from the same species

• Spectral unmixing:

  • Use spectral imaging systems capable of separating overlapping fluorophores

  • Allows more flexibility in fluorophore selection

  • Particularly valuable when analyzing ECI2 colocalization with multiple organelle markers

Example multiplexing panel for studying ECI2 in peroxisome-mitochondria interactions:

• ECI2 Antibody, Biotin conjugated + Streptavidin-Alexa Fluor 488

• Mouse anti-TOMM20 (mitochondrial marker) + Anti-mouse-Alexa Fluor 594

• Goat anti-SCP2 (peroxisomal marker) + Anti-goat-Alexa Fluor 647

This combination allows simultaneous visualization of ECI2 localization relative to both peroxisomes and mitochondria, facilitating analysis of the peroxisome-like structures that mediate organelle contact .

How should quantitative data from experiments using ECI2 Antibody, Biotin conjugated be normalized and analyzed?

Quantitative analysis of ECI2 experiments requires rigorous normalization and statistical approaches:

• Western blot quantification:

  • Normalize ECI2 signal to appropriate loading controls:

    • Total protein (GAPDH) for whole cell lysates

    • Compartment-specific markers for subcellular fractions (COX IV for mitochondria, SCP2 for peroxisomes)

  • Use densitometric analysis software (ImageJ, Image Lab, etc.)

  • Calculate relative distribution ratios between compartments as a measure of localization changes

• Immunofluorescence quantification:

  • Measure colocalization with organelle markers using:

    • Pearson's correlation coefficient

    • Manders' overlap coefficient

    • Object-based colocalization analysis

  • Quantify signal intensity relative to background in defined regions of interest

  • For peroxisome-mitochondria contacts: Measure frequency, duration, and size of contact sites

• Normalization strategies:

  • For cell models: Normalize to cell number, total protein, or housekeeping gene expression

  • For tissue samples: Normalize to tissue weight, total protein, or area of region of interest

  • For subcellular studies: Express data as percentage of total cellular ECI2 in each compartment

• Statistical analysis approaches:

  • For comparing experimental conditions: Apply appropriate statistical tests (t-test, ANOVA)

  • For correlation analysis: Use Pearson's or Spearman's correlation coefficients

  • For time-course experiments: Consider repeated measures ANOVA or mixed models

  • Include biological replicates (n≥3) for robust statistical analysis

• Visualization methods:

  • Present western blot data as bar graphs with error bars

  • For subcellular distribution: Use stacked bar charts showing relative proportions in each compartment

  • For colocalization: Consider heat maps or scatter plots of correlation coefficients

These quantitative approaches can help establish how ECI2 distribution changes during cellular processes and how these changes correlate with metabolic functions like steroid biosynthesis or fatty acid metabolism .

How can I distinguish between specific ECI2 staining and background artifact in immunostaining experiments?

Distinguishing specific ECI2 staining from background artifacts requires systematic controls and careful image analysis:

• Validation through multiple controls:

  • Compare with negative controls (no primary antibody, isotype control)

  • Examine tissues/cells known to express low levels of ECI2

  • Evaluate signal reduction following ECI2 knockdown or blocking peptide competition

  • Cross-validate with non-biotinylated ECI2 antibody

• Subcellular localization assessment:

  • True ECI2 signal should show enrichment in peroxisomes and mitochondria

  • Colocalization with established markers (SCP2 for peroxisomes, COX IV for mitochondria)

  • Diffuse cytoplasmic signal without organelle enrichment may indicate non-specific binding

• Signal characteristics analysis:

  • Specific signal typically shows:

    • Consistent pattern across similar cells/regions

    • Definite subcellular localization

    • Signal intensity proportional to expression level

  • Non-specific signal often presents as:

    • Edge artifacts

    • Nuclear or nucleolar staining (ECI2 is not a nuclear protein)

    • Uniform staining across all tissue/cell types regardless of expression level

• Technical considerations:

  • Autofluorescence: Capture images in empty channels to identify inherent tissue fluorescence

  • Endogenous biotin: Compare with samples processed with streptavidin reagent alone

  • Edge artifacts: Examine staining pattern at tissue/cell boundaries

• Advanced imaging approaches:

  • Z-stack analysis: Specific signals maintain consistent pattern through z-planes

  • Spectral imaging: Can help separate specific signal from autofluorescence

  • Super-resolution microscopy: Enables more precise localization to subcellular structures

Research has shown that ECI2/ACBD2 localizes to both peroxisomes and mitochondria, with specific enrichment at contact sites between these organelles . Any staining pattern that deviates significantly from this expected distribution should be carefully scrutinized for potential artifacts.

What patterns of ECI2 localization should be expected in different cell types and experimental conditions?

ECI2 localization patterns vary by cell type and experimental condition, reflecting its dynamic role in cellular metabolism:

• Baseline distribution in metabolically active cells:

  • Dual localization to peroxisomes and mitochondria

  • Enrichment at peroxisome-mitochondria contact sites

  • Presence in "peroxisome-like structures" that mediate organelle interaction

  • The proximity between peroxisomal ACBD2/ECI2 and mitochondrial TOMM20 has been validated using in-cell co-immunoprecipitation methodology

• Cell type-specific patterns:

  • Steroidogenic cells (e.g., Leydig cells): High expression with prominent localization at organelle contact sites

  • Hepatocytes: Abundant peroxisomal and mitochondrial localization reflecting active fatty acid metabolism

  • Muscle cells: Predominant mitochondrial localization

  • Adipocytes: Dynamic redistribution correlating with lipid metabolism status

• Responses to metabolic stimuli:

  • Hormone stimulation: Increased peroxisome-mitochondria contacts mediated by ECI2

  • Fasting/feeding cycles: Potential redistribution reflecting changes in fatty acid metabolism

  • Ectopic expression of ACBD2/ECI2 isoform A leads to increased basal and hormone-stimulated steroid formation

• Pathological conditions:

  • Metabolic disorders: Potential alterations in ECI2 distribution reflecting perturbed organelle interactions

  • Cellular stress: Possible redistribution as part of adaptive metabolic responses

  • Tissue inflammation: May exhibit altered patterns due to metabolic reprogramming

• Isoform-specific localization:

  • Different ECI2 isoforms may show distinct localization preferences

  • Isoform A has been specifically linked to increased steroid formation

When analyzing ECI2 localization patterns, it's important to consider the specific cellular context and metabolic state. Quantitative assessment of colocalization with organelle markers provides more objective evaluation of distribution changes under different experimental conditions.

What statistical approaches are most appropriate for analyzing colocalization of ECI2 with organelle markers?

When analyzing ECI2 localization, combining multiple statistical approaches provides the most comprehensive assessment of its distribution between peroxisomes and mitochondria, particularly at contact sites between these organelles .

What are the key considerations when designing a comprehensive research strategy using ECI2 Antibody, Biotin conjugated?

Developing a robust research strategy with ECI2 Antibody, Biotin conjugated requires integration of multiple methodological approaches and careful experimental design:

• Validation foundation:

  • Establish antibody specificity through multiple control experiments

  • Confirm expected subcellular localization pattern in well-characterized cell types

  • Cross-validate with non-biotinylated antibodies targeting the same epitope

• Multi-method approach:

  • Combine biochemical techniques (western blotting, immunoprecipitation) with imaging approaches

  • Integrate quantitative and qualitative assessments

  • Correlate localization data with functional readouts of fatty acid metabolism and steroid biosynthesis

  • Research has demonstrated that ECI2/ACBD2 mediates peroxisome-mitochondria interactions that support metabolite exchange for steroid biosynthesis

• Biological context consideration:

  • Select appropriate cell/tissue models based on ECI2 expression levels and biological relevance

  • Steroidogenic cells like MA-10 and mLTC-1 mouse tumor Leydig cells have proven valuable for studying ECI2 function

  • Consider metabolic state and potential dynamic changes in ECI2 distribution

• Technical optimization:

  • Tailor protocol parameters to specific applications (antibody dilution, incubation conditions)

  • Implement appropriate signal amplification strategies for detecting low-abundance signals

  • Address potential challenges like endogenous biotin or high background

• Data integration framework:

  • Develop systematic approaches for integrating results across different experimental platforms

  • Implement quantitative analysis methods for objective assessment

  • Build conceptual models connecting ECI2 localization to its functional roles