EPB41L2 Antibody

What is EPB41L2 and why is it an important research target?

EPB41L2, also known as 4.1G, is a member of the protein 4.1 family that serves as an adaptor linking transmembrane proteins to the cytoskeleton . It plays a critical role in cell structure maintenance and is required for dynein-dynactin complex and NUMA1 recruitment at the mitotic cell cortex during anaphase . Its involvement in maintaining cell shape, integrity, and regulating cellular processes such as cell adhesion and signaling makes it an important target for research into diseases like cancer, where alterations in cell structure and function can contribute to tumor development .

What applications are EPB41L2 antibodies validated for?

EPB41L2 antibodies have been validated for multiple applications including:

The actual working concentration varies and should be determined by the researcher for each specific experimental context .

What are the typical molecular weights observed for EPB41L2 in Western blot applications?

While the calculated molecular weight of EPB41L2 is approximately 112-113 kDa, researchers frequently observe bands at higher molecular weights:

Some researchers have also reported additional bands at approximately 40 kDa in human cerebellum lysates . This discrepancy between calculated and observed molecular weights is likely due to post-translational modifications such as phosphorylation .

How should I optimize antigen retrieval for EPB41L2 immunohistochemistry?

For optimal antigen retrieval in EPB41L2 immunohistochemistry, consider the following approaches:

• Primary recommendation: Use TE buffer pH 9.0 for heat-mediated antigen retrieval. This has been validated on human ovary tumor tissue .

• Alternative approach: If suboptimal results are obtained with TE buffer, citrate buffer pH 6.0 can be used as an alternative .

• Optimization strategy: Perform a side-by-side comparison using both retrieval methods on serial sections of your tissue of interest. Evaluate signal intensity, background staining, and tissue morphology preservation to determine the optimal condition for your specific sample .

For paraffin-embedded tissues, heat-mediated antigen retrieval should be performed before commencing with the IHC staining protocol .

What positive controls are recommended for validating EPB41L2 antibodies?

Based on validated research applications, the following positive controls are recommended:

For human samples, U-87MG, HeLa, Jurkat, and SKOV3 cell lines have been consistently identified as positive for EPB41L2 expression .

How do I troubleshoot non-specific bands in Western blot using EPB41L2 antibodies?

When encountering non-specific bands in Western blot using EPB41L2 antibodies, implement the following troubleshooting strategy:

• Optimize antibody concentration: Test a dilution series (e.g., 1:500, 1:1000, 1:2000) to identify the optimal antibody concentration that provides specific signal with minimal background .

• Adjust blocking conditions: Increase blocking time or concentration (5% milk in TBST is commonly used for EPB41L2 detection) .

• Validate specificity: Consider using blocking peptides corresponding to the immunogen. For instance, bands at approximately 110 kDa and 40 kDa in human cerebellum lysates were successfully blocked by incubation with the immunizing peptide, confirming specificity .

• Consider sample preparation: Ensure complete protein denaturation and use fresh samples to prevent proteolytic degradation that may lead to additional bands.

• Perform knockdown validation: Use siRNA knockdown of EPB41L2 to confirm the specificity of the observed bands, particularly when working with new cell lines or tissues .

How can EPB41L2 antibodies be utilized in studying mitotic cell cortex dynamics?

EPB41L2 is required for dynein-dynactin complex and NUMA1 recruitment at the mitotic cell cortex during anaphase . To study this process:

• Synchronized cell populations: Use a double thymidine block or nocodazole treatment followed by release to enrich for mitotic cells.

• Multi-color immunofluorescence: Combine EPB41L2 antibody (dilution 1:50-1:500) with antibodies against other mitotic markers (e.g., phospho-histone H3) and cortical components (e.g., dynein, dynactin, NUMA1).

• Live-cell imaging: For dynamic studies, consider expressing a fluorescently tagged EPB41L2 construct alongside the immunostaining approach to validate antibody specificity and track protein dynamics in real-time.

• Super-resolution microscopy: Techniques such as STED or STORM can provide enhanced spatial resolution of EPB41L2 localization at the cell cortex during mitosis.

• Proximity labeling: BioID or APEX2 fusion proteins can be used to identify novel EPB41L2 interaction partners at the mitotic cortex, with antibody validation by immunoprecipitation .

What are the considerations for using EPB41L2 antibodies in neuronal tissue research?

When using EPB41L2 antibodies in neuronal tissue research, consider:

• Isoform specificity: EPB41L2 has multiple isoforms (NP_001422.1, NP_001129026.1, NP_001186317.1, etc.) that may be expressed differentially in neuronal tissues. Select antibodies that recognize regions common to all isoforms or specific to your isoform of interest .

• Cross-reactivity: When working with mouse or rat models, confirm the antibody's cross-reactivity with these species. Several antibodies have been validated for reactivity with human, mouse, and rat samples .

• Co-localization studies: EPB41L2 has been implicated in kainate receptor G-protein signaling. Consider co-staining with kainate receptor subunits to examine their relationship in neuronal compartments .

• Blood-brain barrier considerations: For in vivo applications, consider the blood-brain barrier penetration of the antibody if attempting to use it for in vivo imaging or therapeutic targeting.

• Fixation optimization: Neuronal tissues may require specific fixation protocols. Both 4% paraformaldehyde and methanol fixation have been used successfully with EPB41L2 antibodies in various neuronal preparations .

How can EPB41L2 antibodies be employed in studying protein-protein interactions at the membrane-cytoskeleton interface?

To investigate EPB41L2's role as an adaptor linking transmembrane proteins to the cytoskeleton:

• Co-immunoprecipitation: Use EPB41L2 antibodies (0.5-4.0 μg for 1.0-3.0 mg of total protein lysate) for immunoprecipitation followed by mass spectrometry or Western blot detection of interacting partners .

• Proximity ligation assay (PLA): Combine EPB41L2 antibodies with antibodies against suspected interaction partners to visualize molecular proximity (<40 nm) in situ.

• Sucrose gradient fractionation: Use EPB41L2 antibodies to probe different membrane fractions to determine its distribution across membrane microdomains.

• Cross-linking studies: Employ chemical cross-linkers followed by immunoprecipitation with EPB41L2 antibodies to capture transient or weak interactions.

• FRET microscopy: When combined with appropriately labeled secondary antibodies, FRET can provide evidence of direct molecular interactions in fixed cells.

Recent research has identified SLC3A2 and LRBA as main partners of super conserved receptors expressed in the brain that interact with EPB41L2, demonstrating the value of this approach .

How do I validate the specificity of EPB41L2 antibodies for my research application?

To rigorously validate EPB41L2 antibody specificity:

• Positive and negative controls: Include known positive samples (e.g., HeLa cells, Jurkat cells) and negative controls (e.g., cells with EPB41L2 knockdown or knockout) in your experiments .

• Peptide competition assay: Pre-incubate the antibody with blocking peptide corresponding to the immunogen sequence to confirm signal specificity. Several vendors offer matching blocking peptides for their EPB41L2 antibodies .

• Multiple antibody validation: Use at least two different antibodies targeting distinct epitopes of EPB41L2 to confirm consistent localization or expression patterns.

• Orthogonal methods: Validate protein expression using methods that don't rely on antibodies, such as mRNA expression analysis (RT-PCR or RNA-seq).

• Knockout/knockdown validation: Compare signal in wild-type cells versus those where EPB41L2 has been depleted by CRISPR/Cas9 or RNAi approaches .

What are the optimal storage conditions for maintaining EPB41L2 antibody activity?

To maintain optimal EPB41L2 antibody activity over time:

• Storage temperature: Store antibodies at -20°C for long-term storage. Most EPB41L2 antibodies are stable for one year after shipment when stored properly .

• Aliquoting: Upon receipt, divide the antibody into small working aliquots to avoid repeated freeze-thaw cycles. For antibodies stored in glycerol buffer (e.g., PBS with 50% glycerol), aliquoting may be unnecessary for -20°C storage .

• Short-term storage: For frequent use within one month, store at 4°C to avoid repeated freeze-thaw cycles .

• Buffer conditions: Most EPB41L2 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3. Maintain these conditions when diluting the antibody .

• Contamination prevention: Use sterile technique when handling antibodies to prevent microbial contamination, which can lead to degradation.

• Monitoring degradation: If reduced activity is observed over time, run a small amount on SDS-PAGE to check for fragmentation or aggregation .

How should I approach optimization of EPB41L2 antibodies for multi-color immunofluorescence?

For successful multi-color immunofluorescence using EPB41L2 antibodies:

• Host species selection: Choose primary antibodies raised in different host species to enable simultaneous detection. EPB41L2 antibodies are available from rabbit, goat, and mouse hosts .

• Epitope targeting: When using multiple antibodies against EPB41L2, select those targeting different epitopes to avoid competitive binding:

  • N-terminal region (amino acids 1-210)

  • Internal region (amino acids 100-150)

  • C-terminal region (amino acids 593-604)

• Dilution optimization: Perform titration experiments (e.g., 1:50, 1:100, 1:200, 1:500) for each antibody to determine the optimal concentration that provides specific signal with minimal background .

• Sequential immunostaining: If cross-reactivity is observed, consider sequential staining protocols with thorough washing steps between primary-secondary antibody pairs.

• Fluorophore selection: Choose fluorophores with minimal spectral overlap, and when overlap is unavoidable, apply appropriate compensation during image acquisition and analysis.

• Validation in single-color experiments: Before combining antibodies, validate each individually to ensure specific staining patterns .

How do I account for the discrepancy between calculated and observed molecular weights of EPB41L2?

The calculated molecular weight of EPB41L2 is approximately 112-113 kDa, but the observed molecular weight in Western blot is often 130-150 kDa . To account for this discrepancy:

• Post-translational modifications: EPB41L2 undergoes phosphorylation and possibly other modifications that can increase its apparent molecular weight. Consider using phosphatase treatment of lysates to determine if phosphorylation contributes to the shift.

• Splice variants: EPB41L2 has multiple isoforms with different molecular weights. Consider the specific isoform(s) expressed in your experimental system:

  • Isoform a: NP_001422.1

  • Isoform b: NP_001129026.1

  • Isoform c: NP_001186317.1

  • Additional isoforms: NP_001239589.1, NP_001337228.1, etc.

• Protein structure: Highly charged or structured regions can affect protein migration in SDS-PAGE. Consider using different percentage gels or alternative electrophoresis conditions.

• Molecular weight markers: Use reliable molecular weight markers and consider running purified recombinant EPB41L2 as a reference standard.

• Alternative confirmation: Consider mass spectrometry analysis of immunoprecipitated EPB41L2 to accurately determine its molecular weight and identify post-translational modifications .

How should I interpret variations in EPB41L2 subcellular localization across different cell types?

EPB41L2 subcellular localization can vary across cell types, reflecting its diverse functions:

• Membrane vs. cytoskeletal localization: EPB41L2 serves as an adaptor linking transmembrane proteins to the cytoskeleton. The relative distribution between these compartments may vary based on cell type and physiological state .

• Cell junction enrichment: Strong expression has been observed in U2OS cell junctions, suggesting a role in cell-cell adhesion. Compare with established junction markers (e.g., E-cadherin, ZO-1) to confirm co-localization .

• Cortical enrichment during mitosis: EPB41L2 is recruited to the mitotic cell cortex during anaphase. Time-course experiments during the cell cycle can reveal dynamic relocalization .

• Cell-type specific patterns: Systematically document localization patterns across cell types to identify correlations with cellular functions or differentiation states.

• Quantitative approach: Use quantitative image analysis to measure the relative distribution of EPB41L2 between cellular compartments using markers for the cell membrane (e.g., Na+/K+ ATPase), cytoskeleton (e.g., tubulin, actin), and nucleus (DAPI) .

What are the considerations when comparing EPB41L2 expression levels across different experimental conditions?

When comparing EPB41L2 expression across experimental conditions:

• Loading controls: Use appropriate loading controls for normalization. For whole-cell lysates, GAPDH or β-actin are commonly used. For membrane fractions, Na+/K+ ATPase or cadherin may be more appropriate.

• Isoform-specific detection: EPB41L2 has multiple isoforms that may be differentially regulated. Consider using isoform-specific antibodies or RT-PCR to distinguish between them .

• Quantification methods: Use densitometry software to quantify band intensity in Western blots. Report the ratio of EPB41L2 to loading control rather than absolute values.

• Statistical analysis: Perform experiments in biological triplicates at minimum. Apply appropriate statistical tests (e.g., t-test, ANOVA) based on your experimental design.

• Cross-validation: Confirm protein-level changes with mRNA-level analysis (RT-qPCR) when possible, particularly when studying transcriptional regulation.

• Technical considerations: Be aware that different antibodies may preferentially recognize certain post-translational modifications or isoforms, potentially affecting your interpretation of expression changes .

How can EPB41L2 antibodies be employed in super-resolution microscopy studies?

For super-resolution microscopy studies of EPB41L2:

• Antibody selection: Choose antibodies with high specificity and affinity. Monoclonal antibodies like anti-EPB41L2 [EPR8873(2)] (ab175928) may provide more consistent results than polyclonal antibodies .

• Fixation optimization: Test different fixation protocols (4% paraformaldehyde, methanol, or glutaraldehyde) to preserve structure while maintaining epitope accessibility.

• Secondary antibody selection: For STORM or PALM, use secondary antibodies conjugated to photoswitchable fluorophores. For STED, use secondary antibodies with STED-compatible dyes.

• Sample mounting: Use specialized mounting media that reduce photobleaching and improve photoswitching properties for techniques like STORM.

• Resolution calibration: Include fiducial markers to assess the achieved resolution and correct for drift during image acquisition.

• Quantitative analysis: Develop analysis workflows to extract quantitative information about EPB41L2 nanoscale organization, such as cluster size, density, and co-localization with other proteins at the nanometer scale .

What strategies can be employed to study EPB41L2 in patient-derived samples for translational research?

For translational research using patient-derived samples:

• Sample preservation: Optimize tissue preservation protocols (flash freezing or formalin fixation) to maintain EPB41L2 epitope integrity. For IHC applications, heat-mediated antigen retrieval with TE buffer pH 9.0 is recommended .

• Antibody validation in human tissues: Validate antibodies specifically on human tissues. EPB41L2 antibodies have been successfully used on human ovary tumor tissue, uterus, and colon .

• Multi-marker panels: Combine EPB41L2 staining with diagnostic or prognostic markers relevant to the disease context to enhance the informational value.

• Digital pathology: Use whole slide imaging and automated quantification to assess EPB41L2 expression patterns across large patient cohorts.

• Patient-derived organoids/xenografts: Establish models from patient samples to study EPB41L2 function in a more controlled environment while maintaining patient-specific characteristics.

• Correlation with clinical data: Analyze associations between EPB41L2 expression patterns and clinical parameters such as disease progression, treatment response, or survival outcomes .

How can EPB41L2 antibodies be integrated into multiplexed protein detection systems?

For multiplexed protein detection systems incorporating EPB41L2 antibodies:

• Cyclic immunofluorescence: Use EPB41L2 antibodies in cyclic immunofluorescence protocols where antibodies are applied, imaged, and stripped in sequential rounds to build highly multiplexed datasets.

• Mass cytometry (CyTOF): Conjugate EPB41L2 antibodies with rare earth metals for use in mass cytometry, enabling simultaneous detection of dozens of proteins without spectral overlap concerns.

• Multiplex IHC/IF platforms: Incorporate EPB41L2 antibodies into commercial multiplexing platforms like CODEX, Vectra, or GeoMx DSP, following manufacturer guidelines for antibody validation and optimization.

• Antibody conjugation strategies: Consider direct conjugation of EPB41L2 antibodies to oligonucleotides (for CITE-seq or similar approaches) or fluorophores to reduce species cross-reactivity in multiplexed panels.

• Spatial transcriptomics integration: Combine EPB41L2 protein detection with spatial transcriptomics to correlate protein levels with mRNA expression in a spatially resolved manner.

• Validation controls: Include appropriate single-stain controls to assess antibody performance in the multiplexed context and to enable computational correction of spillover or cross-reactivity .