EPS8L3 Antibody, Biotin conjugated

What is EPS8L3 and why is it significant in research?

EPS8L3 (Epidermal growth factor receptor kinase substrate 8-like protein 3) is a protein related to epidermal growth factor receptor pathway substrate 8 (EPS8), functioning as a substrate for the epidermal growth factor receptor. Though its precise function remains largely unknown, it is implicated in EGFR signaling pathways, making it an important research target for cellular signaling studies . The protein exists in multiple isoforms due to alternative splicing, which adds complexity to its study. Research suggests potential roles in cytoskeletal organization and receptor trafficking, similar to other EPS8 family members, though specific functions require further elucidation.

What are the key specifications of biotin-conjugated EPS8L3 antibodies?

Biotin-conjugated EPS8L3 antibodies are polyclonal antibodies typically derived from rabbit hosts with specificity for human EPS8L3. Key specifications include:

The biotin conjugation provides enhanced detection capabilities through avidin-biotin interactions, offering increased sensitivity in various immunological assays .

How should researchers optimize ELISA protocols with biotin-conjugated EPS8L3 antibodies?

For optimal ELISA performance with biotin-conjugated EPS8L3 antibodies, researchers should implement a systematic optimization approach:

• Antibody titration: Begin with manufacturer-recommended dilutions (typically 1:1000 to 1:10000) and perform serial dilutions to determine optimal concentration .

• Blocking optimization: Test different blocking agents (BSA, casein, normal serum) at 2-5% concentrations to reduce background while maintaining specific signal.

• Incubation conditions: Evaluate different incubation times (1-4 hours) and temperatures (room temperature vs. 4°C) for both primary binding and detection steps.

• Detection system selection: Since the antibody is biotin-conjugated, use streptavidin-HRP systems with optimal dilution (typically 1:2000 to 1:10000).

• Signal development: Optimize substrate exposure time (typically 5-30 minutes) using time-course experiments to achieve maximum signal-to-noise ratio.

Researchers should validate results with appropriate positive and negative controls, including recombinant EPS8L3 protein and cell lysates with confirmed EPS8L3 expression patterns .

What cross-reactivity concerns exist when using EPS8L3 antibodies in multi-protein detection systems?

When incorporating biotin-conjugated EPS8L3 antibodies in multi-protein detection systems, researchers should address several cross-reactivity considerations:

• EPS8 family cross-reactivity: EPS8L3 shares sequence homology with other EPS8 family members (EPS8, EPS8L1, EPS8L2), potentially causing cross-recognition. Validate specificity using knockout/knockdown systems or competitive binding assays with recombinant family members .

• Endogenous biotin interference: Tissues with high endogenous biotin (liver, kidney, brain) may produce false positive signals. Pre-block samples with streptavidin/avidin before antibody application or use appropriate blocking kits specifically designed for biotin-based detection systems.

• Secondary detection system interactions: When multiplexing, ensure detection systems (streptavidin conjugates) don't cross-react with other primary or secondary antibodies in the experiment.

• Epitope masking: Consider whether target epitopes might be masked due to protein-protein interactions or post-translational modifications in complex samples.

Data from comprehensive validation studies indicate using peptide pre-absorption controls and comparison with non-biotin conjugated antibodies can effectively distinguish specific from non-specific signals .

How can researchers address inconsistent staining patterns when using biotin-conjugated EPS8L3 antibodies in immunohistochemistry?

Inconsistent immunohistochemical staining with biotin-conjugated EPS8L3 antibodies can result from several factors. A systematic troubleshooting approach includes:

• Antigen retrieval optimization: Test multiple retrieval methods (heat-induced at varying pH values 6.0, 8.0, 9.0; enzymatic retrieval with proteinase K or trypsin) to determine optimal epitope exposure conditions.

• Endogenous biotin blocking: Implement specific biotin/avidin blocking steps (sequential avidin and biotin incubation) to minimize background from endogenous biotin, particularly important in biotin-rich tissues.

• Detection system enhancement: For weak signals, employ tyramide signal amplification (TSA) which can increase sensitivity 10-100 fold compared to conventional detection methods.

• Fixation evaluation: Compare results from differently fixed samples (formalin, paraformaldehyde, alcohol) as overfixation can mask epitopes. For previously fixed samples, extend antigen retrieval times.

• Dilution series: Create a comprehensive dilution series (1:50, 1:100, 1:200, 1:500) to identify optimal antibody concentration that balances specific signal and background .

Analysis of staining patterns from 44 normal human tissues examined with anti-EPS8L3 antibodies reveals variations in expression levels and subcellular localization, necessitating careful optimization for each tissue type .

What are the most effective validation strategies to confirm specificity of EPS8L3 antibody signals?

Rigorous validation of biotin-conjugated EPS8L3 antibody specificity requires a multi-faceted approach:

• Genetic validation:

  • Use CRISPR/Cas9-mediated EPS8L3 knockout cell lines as negative controls

  • Compare staining in EPS8L3-overexpressing vs. normal cells

  • Employ siRNA knockdown experiments with quantitative signal analysis

• Biochemical validation:

  • Peptide competition assays using the immunizing peptide (367-521AA region) at increasing concentrations

  • Western blot analysis confirming the expected molecular weight (approximately 66 kDa)

  • Comparison with non-conjugated antibodies against different EPS8L3 epitopes

• Orthogonal method confirmation:

  • Correlate protein detection with mRNA expression by RT-PCR or RNA-seq

  • Use mass spectrometry to confirm immunoprecipitation results

  • Compare localization patterns with GFP-tagged EPS8L3 constructs

• Cross-platform consistency:

  • Verify consistent detection patterns across multiple applications (ELISA, IHC, IF, WB)

  • Document expected vs. observed subcellular localization patterns

Comprehensive antibody validation studies should involve both positive controls (tissues known to express EPS8L3) and negative controls (tissues with minimal expression) to establish a reliable signal-to-noise threshold .

How can biotin-conjugated EPS8L3 antibodies be utilized in multiplexed immunofluorescence studies?

Multiplexed immunofluorescence with biotin-conjugated EPS8L3 antibodies enables simultaneous visualization of multiple proteins in complex biological samples. Implementation strategies include:

• Sequential multiplexing protocol:

  • Apply biotin-conjugated EPS8L3 antibody (1:100-1:500 dilution)

  • Detect with streptavidin-conjugated fluorophore (e.g., streptavidin-Alexa Fluor 488)

  • Perform antibody stripping (glycine buffer pH 2.5 or commercial antibody removal buffer)

  • Block and apply subsequent primary-secondary antibody pairs with non-overlapping fluorophores

  • Document complete signal removal between cycles using no-primary controls

• Spectral unmixing approach:

  • Apply biotin-conjugated EPS8L3 antibody simultaneously with other primary antibodies from different host species

  • Use streptavidin-conjugated fluorophore and species-specific secondary antibodies with distinct emission spectra

  • Employ spectral imaging and linear unmixing algorithms to separate overlapping signals

  • Include single-stained controls for accurate spectral fingerprinting

• Tyramide signal amplification integration:

  • Utilize HRP-streptavidin with tyramide-conjugated fluorophores for signal amplification

  • Perform heat-mediated antibody removal (microwave or pressure cooker treatment)

  • Achieve 5-7 marker multiplexing on a single tissue section

This approach allows correlation of EPS8L3 expression with other proteins in the EGFR signaling pathway or cytoskeletal organization markers, revealing potential co-localization patterns and functional relationships .

What strategies can address epitope masking when investigating post-translational modifications of EPS8L3?

Investigating post-translational modifications (PTMs) of EPS8L3 presents unique challenges due to potential epitope masking. Researchers can implement these advanced strategies:

• Modification-specific sample preparation:

  • For phosphorylation studies: Include phosphatase inhibitors (sodium orthovanadate, sodium fluoride) in all buffers

  • For ubiquitination analysis: Add deubiquitinase inhibitors (PR-619, N-ethylmaleimide)

  • For glycosylation examination: Compare native samples with deglycosylated samples (PNGase F, O-glycosidase treatment)

• Epitope retrieval optimization:

  • Utilize different detergents (NP-40, Triton X-100, SDS) at varying concentrations to expose masked epitopes

  • Test chaotropic agents (urea at 2-4M) for partial protein denaturation

  • Apply heat-mediated retrieval at different pH conditions (citrate pH 6.0 vs. EDTA pH 8.0-9.0)

• Complementary antibody approach:

  • Combine biotin-conjugated EPS8L3 antibody (targeting aa 367-521) with antibodies recognizing different epitopes (aa 1-30 N-terminal or aa 401-450)

  • Use PTM-specific antibodies (anti-phosphotyrosine, anti-ubiquitin) in co-localization studies

  • Employ proximity ligation assays to confirm spatial relationships between EPS8L3 and suspected modifying enzymes

• Mass spectrometry validation:

  • Perform immunoprecipitation with the biotin-conjugated antibody

  • Subject captured proteins to tryptic digestion and LC-MS/MS analysis

  • Map identified PTMs against the EPS8L3 sequence and known modification motifs

These approaches have successfully uncovered functionally significant phosphorylation sites and other modifications in EPS8 family proteins, potentially revealing regulatory mechanisms affecting EPS8L3 activity in signaling cascades .

How does biotin-conjugated EPS8L3 antibody performance compare to unconjugated alternatives in different applications?

A systematic comparison between biotin-conjugated and unconjugated EPS8L3 antibodies reveals application-specific advantages and limitations:

Research data indicates that while biotin-conjugated antibodies offer superior sensitivity in most applications, unconjugated versions provide greater flexibility and reduced background in complex tissue samples. Selection should be guided by specific experimental requirements and target abundance .

What are the critical considerations when designing co-immunoprecipitation experiments with EPS8L3 antibodies?

Successful co-immunoprecipitation (Co-IP) experiments with EPS8L3 antibodies require careful experimental design to preserve protein-protein interactions while achieving specific target capture:

• Lysis buffer optimization:

  • Test mild non-ionic detergents (0.5-1% NP-40, 0.5% Triton X-100) to preserve interactions

  • Adjust salt concentration (150-300mM NaCl) to balance specificity and interaction preservation

  • Include appropriate protease and phosphatase inhibitors (PMSF, aprotinin, leupeptin, sodium orthovanadate)

  • Consider detergent-free extraction methods for membrane-associated complexes

• Capture strategy selection:

  • Direct approach: Use biotin-conjugated EPS8L3 antibody with streptavidin magnetic beads

  • Indirect approach: Use unconjugated EPS8L3 antibody with Protein G beads

  • Pre-clearing protocol: Implement 30-minute pre-clearing with appropriate beads to reduce non-specific binding

• Interaction validation controls:

  • Input control: 5-10% of pre-IP lysate

  • Negative controls: Non-specific IgG and/or immunoprecipitation from EPS8L3-depleted cells

  • Reversed Co-IP: Confirm interaction by immunoprecipitating suspected binding partners

  • Competitive elution: Use immunogen peptide to specifically elute EPS8L3 complexes

• Detection optimization:

  • Western blot detection: Use antibodies against different epitopes than the capture antibody

  • Mass spectrometry: Consider on-bead digestion to identify novel interaction partners

  • Proximity-dependent labeling: Combine with BioID or APEX2 approaches for comprehensive interactome mapping

Research indicates that EPS8L3 interactions with cytoskeletal regulators and receptor trafficking components may be particularly sensitive to detergent conditions. Comparative studies suggest that biotin-conjugated antibodies can provide cleaner Co-IP results when using appropriate blocking to prevent non-specific streptavidin interactions .

How might biotin-conjugated EPS8L3 antibodies contribute to understanding tissue-specific functions of EPS8L3?

Biotin-conjugated EPS8L3 antibodies offer unique advantages for elucidating tissue-specific EPS8L3 functions through several advanced research applications:

• Tissue microarray (TMA) profiling:

  • Apply standardized immunohistochemistry protocols across multi-tissue arrays

  • Quantify expression levels using digital pathology scoring systems

  • Correlate expression patterns with tissue-specific transcriptome data

  • This approach has revealed variable EPS8L3 expression across 44 normal human tissues with notable enrichment in epithelial and glandular tissues

• Single-cell analysis integration:

  • Combine antibody-based protein detection with single-cell RNA sequencing

  • Implement CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) with biotin-conjugated antibodies

  • Map cell type-specific expression patterns within heterogeneous tissues

  • Correlate with function-associated gene modules to predict contextual roles

• Spatial proteomics applications:

  • Utilize multiplexed immunofluorescence with tissue clearing techniques

  • Apply digital spatial profiling with biotin-conjugated antibodies as fiducial markers

  • Generate 3D expression maps correlated with tissue architecture and cellular organization

  • This approach has potential to reveal microenvironment-dependent EPS8L3 functions

• Functional genomics correlation:

  • Integrate antibody-based protein quantification with CRISPR screening data

  • Assess tissue-specific phenotypic consequences of EPS8L3 modulation

  • Map differential protein interaction networks across tissue contexts

These approaches collectively provide a framework for understanding why and how EPS8L3 functions vary across tissue contexts, potentially revealing specialized roles in different cell types and providing insight into its currently undetermined biological functions .

What considerations are important when using EPS8L3 antibodies in studying developmental processes?

Studying developmental processes with EPS8L3 antibodies requires specific considerations to address temporal expression patterns, isoform variations, and developmental context:

• Developmental stage-specific protocol optimization:

  • Adjust fixation protocols based on embryonic/fetal tissue characteristics (reduced fixation times)

  • Modify antigen retrieval conditions for developing tissues (gentler conditions for embryonic samples)

  • Implement tissue-specific blocking strategies (higher serum percentages for embryonic tissues)

  • Test dilution ranges broader than adult tissues (1:50-1:1000) due to potential expression level variations

• Isoform detection strategy:

  • Assess suitability of the epitope region (367-521AA) for detecting developmentally regulated isoforms

  • Compare with antibodies targeting different regions to identify potential isoform switching

  • Correlate with isoform-specific PCR to validate protein-level observations

  • Consider the impact of alternative splicing events on epitope availability

• Contextual validation approach:

  • Implement side-by-side comparisons with other developmental markers

  • Use knockout/knockdown models with stage-specific induction

  • Correlate with known developmental signaling pathways (Wnt, Notch, BMP)

  • Compare with expression patterns of other EPS8 family members to identify potential compensatory mechanisms

• Technical adaptations for developmental contexts:

  • Increase antibody concentration for earlier developmental stages (1:50-1:100)

  • Extend incubation times for better penetration in whole-mount applications

  • Implement more rigorous controls for each developmental timepoint

  • Consider clearing techniques for three-dimensional imaging of intact embryonic structures

Research indicates that EPS8 family proteins show dynamic expression patterns during development, with potential roles in tissue morphogenesis and differentiation. The biotin-conjugated format offers advantages for detecting low-abundance expression in early developmental contexts, potentially revealing previously uncharacterized functions of EPS8L3 in embryonic and fetal development .