EGFLAM Antibody

What is EGFLAM and why is it significant in research applications?

EGFLAM (EGF-like, fibronectin type-III and laminin G-like domain-containing protein), also known as Pikachurin or Nectican, is a highly conserved extracellular matrix-like protein with a molecular weight around 110 kDa. This protein is primarily known for its role in mediating cell-cell and cell-matrix interactions.

EGFLAM is expressed in various organs and tissues including brain, endocrine tissues and muscle tissues . At the cellular level, EGFLAM is colocalized with both dystrophin and dystroglycan at the ribbon synapses, playing critical roles in interactions between the photoreceptor ribbon synapse and bipolar dendrites .

The protein contains two fibronectin type-III domains followed by three EGF-like domains and three Laminin G-like domains. Mouse EGFLAM shares 89% and 95% amino acid identity with human and rat EGFLAM, respectively, indicating high evolutionary conservation .

Research significance of EGFLAM has expanded beyond its structural role to include:

• Biomarker potential in cancer research, particularly glioblastoma

• Role in aortic aneurysm development

• Association with ovarian cancer through hypomethylation patterns

• Potential therapeutic target in various pathological conditions

What types of EGFLAM antibodies are available for research and how do they differ?

Researchers have access to various types of EGFLAM antibodies, each with distinct properties suitable for different experimental applications:

Monoclonal Antibodies:

• Offer high specificity for particular EGFLAM epitopes

• Provide consistent results across experiments

• Available as polyclonal or monoclonal formats

• Some are engineered with tags (e.g., FLAG-tag) to facilitate detection

Polyclonal Antibodies:

• Recognize multiple epitopes on the EGFLAM protein

• Available in formats such as the EGFLAM Antibody (PACO36466) which exhibits:

  • Host species: Rabbit

  • Recommended applications: ELISA (1:2000-1:10000) and Western Blot (1:1000-1:5000)

  • Species reactivity: Human, Mouse

  • Immunogen design: Recombinant Human Pikachurin protein (25-300AA)

Research-grade Antibodies:

• Purified through methods like Protein G purification (>95% purity)

• Available in non-conjugated forms or with fluorescent/enzymatic tags

• Stored in specific buffers (e.g., 50% Glycerol, 0.01M PBS, pH 7.4) with preservatives

The choice between these formats depends on the intended application, required sensitivity, and experimental design considerations.

What are the validated applications for EGFLAM antibodies in laboratory research?

EGFLAM antibodies have been validated for multiple research applications, with specific optimization parameters for each technique:

Western Blotting:

• Successfully detects EGFLAM protein bands at 103-198 kDa under reducing and non-reducing conditions

• Typical working dilutions range from 1:1000-1:5000

• Can detect both recombinant and endogenous EGFLAM from tissues like mouse heart

Enzyme-Linked Immunosorbent Assay (ELISA):

• Working dilutions typically range from 1:2000-1:10000

• Useful for quantitative assessment of EGFLAM expression levels

• Can be adapted for PK bridging assays using anti-idiotypic capture antibody formats

Immunofluorescence and Immunohistochemistry:

• Allows visualization of EGFLAM localization in tissue sections

• Particularly useful for studying EGFLAM at photoreceptor ribbon synapses

• Requires optimization of antigen retrieval methods depending on tissue type

Flow Cytometry:

• Enables quantitative analysis of EGFLAM expression at the cellular level

• Particularly valuable for studying heterogeneous cell populations

FluoroSpot Assays:

• Can be configured for multiplexed detection of EGFLAM alongside other proteins

• Allows for assessment of cross-reactivity and epitope specificity

• Provides reliable and consistent results regardless of assay complexity

Each application requires specific optimization of antibody concentration, incubation conditions, and detection methods for optimal results.

How does EGFLAM expression relate to cancer progression, particularly in glioblastoma?

EGFLAM has emerged as a significant marker in cancer research, particularly in glioblastoma (GBM), with important implications for understanding disease mechanisms and developing targeted therapies:

Expression Pattern in GBM:

• EGFLAM is significantly upregulated in GBM tissues compared to normal brain tissues as demonstrated by analyses of both TCGA dataset (169 GBM tissues, 5 normal brain tissues) and Oncomine database (22 GBM tissues, 3 normal brain tissues)

• Higher EGFLAM expression is associated with poor prognosis in GBM patients

• EGFLAM expression is remarkably associated with GBM subtype (p<0.003) but shows no significant association with age, gender, and Karnofsky Performance Status (KPS)

Cellular Functions in GBM Progression:

• Laboratory investigations demonstrate that EGFLAM significantly promotes proliferation of GBM cells as evidenced by:

  • CCK-8 assays showing reduced cell viability upon EGFLAM knockdown

  • Colony formation assays revealing attenuated colony formation after EGFLAM silencing

• EGFLAM enhances migration and invasion of GBM cells, confirmed through:

  • Wound healing assays showing wider wound width in si-EGFLAM group

  • Transwell assays demonstrating reduced numbers of invading and migrating cells after EGFLAM knockdown

Molecular Mechanisms:

• EGFLAM activates the PI3K/AKT signaling pathway in GBM cells

• Western blot analysis shows decreased expression of p-PI3K, p-AKT, and p-P70S6K in EGFLAM-silenced cells

• EGFLAM doesn't affect total PI3K and AKT levels, suggesting it specifically modulates phosphorylation status

Research Implications:

• EGFLAM antibodies are valuable tools for studying these cancer-related processes

• Immunoprofiling of tumor samples using EGFLAM antibodies may have prognostic value

• EGFLAM may represent a novel therapeutic target for GBM treatment strategies

What methodologies can researchers use to validate the specificity of EGFLAM antibodies?

Ensuring antibody specificity is critical for reliable research outcomes. Several complementary approaches can validate EGFLAM antibody specificity:

Western Blot Validation:

• Verify antibody detects bands of expected molecular weight (approximately 110-112 kDa for full-length EGFLAM)

• Compare detection patterns in tissues known to express EGFLAM (brain, heart) versus negative controls

• Use reducing and non-reducing conditions to assess recognition of structural epitopes

• Analyze bands using Coomassie Blue staining to confirm protein integrity

Recombinant Protein Controls:

• Test antibody against recombinant EGFLAM proteins with defined sequences

• Use tagged recombinant proteins (e.g., Flag-tagged mouse EGFLAM) as positive controls

• Analyze binding affinities with techniques like surface plasmon resonance

Cross-Reactivity Testing:

• Assess reactivity across species (human, mouse) to confirm expected cross-reactivity pattern

• Test against related proteins containing similar domains to ensure specificity

• Perform epitope mapping to determine precise binding regions

FluoroSpot Assay Configuration:

• Implement multiplexed assays using different detection reagents

• Use configurations like 1×1 (single antigen, single detection reagent), 1×4 (single antigen, multiple detection reagents), 4×1 (multiple antigens, single detection reagent), and 4×4 (multiple antigens, multiple detection reagents)

• Compare spot counts across configurations to ensure consistency regardless of complexity

Genetic Approaches:

• Use EGFLAM-knockout models or siRNA-treated cells as negative controls

• Compare antibody reactivity in wild-type versus EGFLAM-knockdown samples

• Perform EGFLAM overexpression experiments to validate increased signal detection

Epitope Analysis:

• Determine whether antibodies recognize linear or conformational epitopes through appropriate sample preparation methods

• Test recognition of EGFLAM fragments to map binding domains

• Consider post-translational modifications that might affect antibody binding

This multi-faceted validation approach ensures robust antibody performance across experimental applications.

How can researchers optimize EGFLAM antibody-based assays for investigating signaling pathways?

Optimizing antibody-based assays for studying EGFLAM's role in signaling requires careful experimental design and methodological considerations:

PI3K/AKT Pathway Analysis:

• EGFLAM has been shown to activate the PI3K/AKT pathway in GBM cells

• For comprehensive pathway analysis, researchers should probe for:

  • Total PI3K and AKT (baseline expression)

  • Phosphorylated forms: p-PI3K, p-AKT, and p-P70S6K (activation status)

  • Downstream effectors specific to the research context

Methodological Optimization:

• Antibody Selection: Choose phospho-specific antibodies with documented specificity

• Sample Preparation: Preserve phosphorylation status through rapid lysis in phosphatase inhibitor-containing buffers

• Signal Enhancement: Implement tyramide signal amplification for weak signals

• Normalization: Use appropriate housekeeping proteins as loading controls

• Quantification: Apply densitometric analysis to quantify relative activation levels

Multiplexed Detection Approaches:

• Implement co-immunoprecipitation to detect EGFLAM-interacting proteins

• Use proximity ligation assays to visualize protein-protein interactions in situ

• Apply phospho-protein arrays for broader pathway analysis

• Consider FluoroSpot assays for detecting multiple targets simultaneously

Functional Validation Strategies:

• Combine EGFLAM antibody-based detection with functional readouts:

  • Cell proliferation assays (CCK-8, colony formation)

  • Migration assays (wound healing)

  • Invasion assays (transwell)

• Include pathway inhibitors (e.g., PI3K inhibitors) as experimental controls

• Correlate signaling pathway activation with functional outcomes

Troubleshooting Pathway Analysis:

• For weak phospho-protein signals: optimize cell starvation and stimulation conditions

• For inconsistent results: standardize lysate preparation and handling procedures

• For high background: increase blocking stringency and optimize antibody dilutions

• For non-specific bands: consider using monoclonal antibodies with higher specificity

This methodological framework provides a comprehensive approach to investigating EGFLAM's role in cell signaling pathways.

What are the key considerations when developing or selecting bispecific antibodies that target EGFLAM?

Bispecific antibodies (BsAbs) targeting EGFLAM represent an advanced frontier in research and potential therapeutic development. Key considerations include:

Molecular Format Selection:

• Different BsAb formats offer distinct advantages for EGFLAM targeting:

  • Dual-variable domain immunoglobulin (DVD-Ig): Provides two binding sites against each antigen, offering higher avidity

  • Knob-in-hole (KIH): Ensures correct pairing with one binding site against each antigen

  • Y-shaped with additional fragments: Can combine full-length antibody with additional binding fragments for enhanced functionality

Target Selection for Bispecificity:

• When EGFLAM is one target, consider complementary secondary targets based on:

  • Coexpressed receptors in disease tissue (e.g., growth factor receptors)

  • Pathway components that synergize with EGFLAM signaling (e.g., PI3K/AKT pathway members)

  • Immune checkpoint molecules for potential immunotherapeutic applications

Epitope Engineering:

• Target specific EGFLAM domains based on functional relevance:

  • EGF-like domains for receptor interactions

  • Laminin G-like domains for extracellular matrix binding

  • Fibronectin type-III domains for cell adhesion functions

• Consider epitope accessibility in the native protein conformation

Analytical Characterization:

• Employ complementary methods to characterize BsAb functionality:

  • Binding kinetics to each target independently and simultaneously

  • Structural analysis through crystallography or cryo-EM

  • Physicochemical characterization (SEC, mass spectrometry)

  • Cross-reactivity assessment with related proteins

Functional Validation:

• Assess BsAb efficacy through:

  • Cell-based assays measuring EGFLAM-dependent functions

  • Pathway inhibition analysis (e.g., PI3K/AKT signaling)

  • In vivo models where EGFLAM plays a role (e.g., GBM xenografts)

  • Comparative analysis with monospecific antibodies targeting each epitope separately

Production and Stability Considerations:

• Evaluate expression systems for optimal yield and quality

• Assess stability under various storage and handling conditions

• Consider glycosylation patterns that may affect function

• Test for aggregation propensity and immunogenicity risk factors

This structured approach helps researchers develop effective bispecific antibodies targeting EGFLAM for advanced research and potential therapeutic applications.

How can researchers effectively troubleshoot common issues in EGFLAM antibody-based experiments?

Experimental challenges with EGFLAM antibodies can compromise research outcomes. Here are systematic troubleshooting approaches for common issues:

Issue: Low or No Signal in Western Blots

Issue: Non-specific Bands or High Background

Issue: Inconsistent Immunostaining Results

Issue: Poor Reproducibility in Functional Assays

Advanced Troubleshooting for Complex Assays:

• For FluoroSpot assays, test simplified configurations (1×1) before advancing to complex multiplexed formats (4×4)

• For cross-reactivity assessment, include epitope mapping to identify binding regions

• For inconsistent results across species, verify epitope conservation through sequence alignment

• For pathway analysis inconsistencies, implement phosphatase inhibitors immediately upon cell lysis

Systematic application of these troubleshooting approaches can resolve most common issues encountered in EGFLAM antibody-based research.