ESAM Antibody

What is ESAM and why is it significant in research?

ESAM is a 55-kDa membrane protein composed of two extracellular immunoglobulin domains, a single transmembrane domain, and a 120 amino acid cytoplasmic domain. It belongs to the junctional adhesion molecule (JAM) family and is primarily expressed on endothelial cells and activated platelets . The significance of ESAM in research stems from its crucial role in regulating vascular permeability in response to factors such as VEGF and during neutrophil extravasation . Additionally, ESAM serves as an effective marker for lifelong hematopoietic stem cells (HSCs) in both mice and humans, making it valuable for developmental and regenerative medicine research .

What are the common applications for ESAM antibodies?

ESAM antibodies are utilized across multiple research applications, with flow cytometry being the most widely reported method. Other common applications include:

• Flow cytometric analysis for identifying ESAM-expressing cells (recommended usage ≤0.25 μg per test)

• Western blotting for protein expression analysis

• Immunohistochemistry for tissue localization studies

• Enzyme-linked immunosorbent assay (ELISA)

• Functional studies examining cell adhesion mechanisms

• Developmental studies investigating hematopoiesis

Flow cytometry applications typically involve excitation at 488-561 nm and emission at 578 nm, using blue, green, or yellow-green lasers .

How does ESAM expression vary across different tissues and cell types?

• Activated platelets show significant ESAM expression

• Hemogenic endothelium in developing aorta exhibits high ESAM expression before definitive HSC establishment

• Expression levels differ substantially between myeloid-erythroid progenitors in the yolk sac and definitive HSCs in intra-embryonic sites

• ESAM can be used as a marker to identify blood vessel endothelial cells

In human tissues, the canonical ESAM protein has a reported length of 390 amino acid residues with a mass of 41.2 kDa, and up to two different isoforms have been reported .

How does ESAM contribute to hematopoietic stem cell development and function?

Research has revealed that ESAM plays crucial roles in definitive hematopoiesis development. Studies using ESAM-knockout mice demonstrated that:

• ESAM deficiency leads to a marked decrease in HSCs with definitive phenotype in fetal livers at E14.5 and is detectable as early as E13.5

• ESAM-null HSCs exhibit functional disruption in differentiation capacity in culture

• The lymphopoietic activity (an authentic feature of definitive HSCs) is impaired in ESAM-null HSCs

• ESAM-null HSCs show decreased frequencies of progenitors with lymphopoietic potential by approximately 40% in the LSK CD48- fraction of ESAM-null fetal livers

• While ESAM-null HSCs can reconstitute hematopoiesis in wild-type mice, they demonstrate reduced adult-type hemoglobin synthesis ability

These findings suggest that ESAM is functionally involved in the development of definitive HSCs and adult-type erythropoiesis .

What molecular signaling mechanisms does ESAM participate in?

ESAM participates in several significant molecular signaling pathways that impact cell function:

• Through homophilic binding, ESAM expressed on HSCs can transduce signals that affect gene expression profiles, particularly erythropoiesis-related genes

• Crosslinking of ESAM with anti-ESAM antibodies affects expression of numerous genes - upregulating 365 genes and downregulating 358 genes

• Pathway network analysis indicates that ESAM signaling influences hematological system development and function, hematocrit levels, and molecular transport networks associated with hemoglobin synthesis

• At endothelial tight junctions, ESAM participates in the regulation of vascular permeability in response to agents such as VEGF or during neutrophil extravasation

Understanding these signaling mechanisms provides insights into how ESAM antibodies might be used to manipulate these pathways in experimental settings.

What are the consequences of ESAM deficiency in experimental models?

ESAM knockout and conditional knockout mouse models have revealed important phenotypes:

• Life-threatening hematopoietic events occur after E15.5 in ESAM-null mice

• ESAM-null HSCs demonstrate reduced expression of important erythropoiesis genes including Hba, Hbb-b1, and Alas2 in HSC-derived BFU-E colonies

• Recipients of transplanted ESAM-null HSCs show significantly lower hemoglobin levels in peripheral blood despite normal red blood cell counts

• While engraftment and proliferation abilities of HSCs and progenitor cells are not disrupted by ESAM deficiency, the maintenance of adult-type hemoglobin synthesis is compromised

• B-cell output from ESAM-null HSCs is significantly lower than wild-type when cocultured with supportive stromal cells, although myeloid cell growth remains equivalent

These findings suggest that ESAM plays specialized roles in certain hematopoietic lineages, particularly in erythroid and lymphoid development.

What are the key factors in selecting the appropriate ESAM antibody for specific applications?

When selecting an ESAM antibody for research, consider these critical factors:

• Application compatibility: Verify the antibody has been validated for your specific application (flow cytometry, immunohistochemistry, Western blotting, etc.)

• Species reactivity: Ensure the antibody recognizes your species of interest (ESAM orthologs exist in mouse, rat, bovine, frog, chimpanzee, and chicken)

• Clone type: Determine whether monoclonal (like clone 1G8) or polyclonal antibodies are more suitable for your application

• Conjugation: Select appropriate fluorophores for flow cytometry (PE-conjugated antibodies have excitation at 488-561 nm and emission at 578 nm)

• Epitope specificity: Confirm which region of ESAM the antibody recognizes (e.g., extracellular domain Gln30-Ser248)

• Supporting validation data: Review available data demonstrating antibody specificity and performance in relevant applications

For quantitative assessments, signal-to-noise ratio and dynamic range are crucial parameters to evaluate when selecting antibodies .

How should researchers optimize ESAM antibody concentration for different experimental protocols?

Optimizing antibody concentration is essential for reliable results. The following methodological approach is recommended:

• For flow cytometry: Begin with ≤0.25 μg per test in a final volume of 100 μL, adjusting cell numbers empirically (typically between 10^5 to 10^8 cells/test)

• For immunohistochemistry: Start with approximately 5 μg/ml as used in mouse tissue studies with anti-ESAM antibodies

• Perform titration experiments to determine the optimal antibody concentration that maximizes specific signal while minimizing background:

  • Too much antibody can yield nonspecific results

  • Too little can lead to false-negative results or no data

• Follow vendor recommendations for protein-specific antigen retrieval methods

• If results are suboptimal, adjust both retrieval methods and corresponding antibody concentration accordingly

• Document optimal conditions for reproducibility across experiments

This systematic approach ensures consistent and reliable experimental outcomes.

What technical challenges might researchers encounter when using ESAM antibodies?

Several technical challenges may arise when working with ESAM antibodies:

• Post-translational modifications: ESAM undergoes glycosylation, which may affect antibody recognition depending on the epitope

• Isoform specificity: Up to two different ESAM isoforms have been reported in humans, requiring careful antibody selection to target the isoform of interest

• Tissue-specific expression levels: Expression varies across tissues and developmental stages, necessitating sensitive detection methods

• Cross-reactivity: Antibodies may cross-react with related JAM family members due to structural similarities

• Fixation sensitivity: Some epitopes may be affected by fixation methods, requiring optimization of sample preparation protocols

• Antigen retrieval: For IHC applications, heat-induced epitope retrieval using basic buffers (such as VisUCyte Antigen Retrieval Reagent-Basic) may be necessary to unmask antigenic sites

Understanding these challenges allows researchers to implement appropriate controls and optimization strategies.

What are the essential validation steps for confirming ESAM antibody specificity?

Proper validation is critical for ensuring antibody specificity and experimental reproducibility. Key validation steps include:

• Testing for specificity, sensitivity, and reproducibility across multiple experimental runs

• Including positive control samples with known ESAM expression (e.g., endothelial cell lines like bEnd.3)

• Using negative controls that lack ESAM expression to confirm absence of non-specific binding

• Implementing isotype controls (such as AB-108-C) to distinguish specific from non-specific binding in flow cytometry

• Utilizing knockout or knockdown models when available to confirm antibody specificity

• Employing multiple antibodies targeting different epitopes of ESAM to cross-validate findings

• Verifying expected subcellular localization (membrane localization for ESAM)

• Confirming expected molecular weight (55 kDa in mouse, 41.2 kDa for canonical human protein) in Western blot applications

These validation steps help ensure that observed signals genuinely represent ESAM rather than non-specific interactions.

What controls should be included in ESAM antibody experiments?

Every experiment utilizing ESAM antibodies should include appropriate controls:

• Positive controls: Samples with verified ESAM expression, such as endothelial cells or activated platelets

• Negative controls: Samples lacking ESAM expression or isotype-matched control antibodies

• Expression gradient controls: A series of samples with variable ESAM expression levels to demonstrate antibody sensitivity and dynamic range

• Tissue microarrays (TMAs): For IHC applications, TMAs containing various tissue samples can serve as quality control and reproducibility references

• Cell line arrays: Arrays of cell lines with a range of ESAM expression levels can be run alongside experiments

• Secondary antibody-only controls: To detect non-specific binding of secondary detection reagents

• For functional studies, appropriate experimental controls such as crosslinking with isotype-matched control antibodies should be included

These controls should be run with every experiment to ensure reliable and reproducible results.

How can researchers troubleshoot problems with ESAM antibody staining?

When encountering issues with ESAM antibody staining, consider these methodological troubleshooting approaches:

• For weak or absent signal:

  • Increase antibody concentration within recommended ranges

  • Optimize antigen retrieval methods (for IHC applications)

  • Extend incubation time with primary antibody

  • Verify sample preparation and fixation procedures

  • Confirm ESAM expression in the sample using alternative methods

• For high background or non-specific staining:

  • Decrease antibody concentration

  • Include additional blocking steps

  • Optimize washing procedures

  • Use more specific detection systems

  • Verify secondary antibody compatibility

• For inconsistent results:

  • Standardize sample preparation protocols

  • Document lot numbers and storage conditions of antibodies

  • Prepare fresh working solutions of antibodies

  • Maintain consistent incubation times and temperatures

  • Include internal controls with every experiment

• For flow cytometry applications:

  • Adjust compensation settings for multicolor panels

  • Optimize sample preparation to maintain ESAM epitope integrity

  • Use viability dyes to exclude dead cells that may bind antibodies non-specifically

Systematic troubleshooting using these approaches can help identify and resolve technical issues with ESAM antibody staining.

How can ESAM antibodies be utilized in hematopoietic stem cell research?

ESAM antibodies offer powerful tools for HSC research applications:

• Identification and isolation of definitive HSCs: ESAM serves as an effective marker for lifelong HSCs in both mice and humans

• Developmental studies: ESAM expression can be used to track the emergence of definitive hematopoiesis during embryonic development

• Functional manipulation: Crosslinking ESAM with antibodies can influence gene expression profiles in HSCs, particularly affecting erythropoiesis-related genes

• Lineage tracing: ESAM expression patterns can help distinguish between myeloid-erythroid progenitors in the yolk sac and definitive HSCs in intra-embryonic sites

• Transplantation studies: ESAM antibodies can be used to evaluate HSC engraftment and reconstitution potential

• Signaling pathway analysis: Anti-ESAM antibodies can help elucidate the molecular mechanisms by which ESAM influences HSC development and function

These applications provide valuable insights into the fundamental biology of HSCs and their developmental origins.

What approaches can be used to study ESAM's role in endothelial cell function?

To investigate ESAM's functions in endothelial cells, researchers can employ several methodological approaches:

• Immunohistochemical analysis of vascular endothelial cells in tissues such as kidney and liver to examine ESAM localization to cell junctions

• Permeability assays using endothelial monolayers and ESAM antibodies or knockdown/knockout models to assess barrier function

• Flow cytometry to quantify ESAM expression levels on endothelial cells under various conditions (e.g., inflammatory stimuli)

• Co-immunoprecipitation studies to identify ESAM-interacting proteins at endothelial tight junctions

• Live-cell imaging with labeled ESAM antibodies to track dynamics during processes such as neutrophil extravasation

• Functional blocking studies using ESAM antibodies to disrupt homophilic interactions and assess effects on endothelial barrier integrity

These approaches can reveal how ESAM contributes to endothelial cell function in normal physiology and disease states.

How can researchers integrate ESAM antibodies into multi-parameter flow cytometry panels?

Designing effective multi-parameter flow cytometry panels incorporating ESAM antibodies requires careful consideration:

• Fluorophore selection: PE-conjugated anti-ESAM antibodies (excitation: 488-561 nm; emission: 578 nm) can be used with blue, green, or yellow-green lasers

• Panel design: Use tools like the Invitrogen Flow Cytometry Panel Builder to integrate ESAM antibodies with other markers of interest

• Titration optimization: Carefully titrate the ESAM antibody (recommended ≤0.25 μg per test) to determine optimal concentration for specific signal

• Compensation controls: Include single-stained controls for each fluorophore in the panel to enable proper compensation

• FMO (Fluorescence Minus One) controls: Include FMO controls to set accurate gates, especially for markers with continuous expression patterns

• Viability discrimination: Include viability dyes to exclude dead cells that may bind antibodies non-specifically

• Sample preparation standardization: Maintain consistent cell numbers (10^5 to 10^8 cells/test) and staining volumes (typically 100 μL)

These methodological considerations ensure robust and reproducible multi-parameter flow cytometry data incorporating ESAM detection.

What emerging applications might expand the utility of ESAM antibodies?

ESAM antibody applications continue to evolve, with several promising research directions:

• Single-cell analysis: Integration of ESAM antibodies into single-cell RNA-seq workflows to correlate protein expression with transcriptional profiles

• Spatial transcriptomics: Combining ESAM immunodetection with spatial transcriptomics to map expression patterns in tissue contexts

• Therapeutic targeting: Development of function-blocking ESAM antibodies to modulate vascular permeability in pathological conditions

• Regenerative medicine: Using ESAM as a marker for HSC identification and isolation in stem cell therapy applications

• Developmental biology: Further exploration of ESAM's role in the transition from primitive to definitive hematopoiesis

• Disease models: Investigation of ESAM expression and function in pathological conditions affecting vascular integrity and hematopoiesis

These emerging applications highlight the continued importance of well-validated ESAM antibodies in biomedical research.

What methodological advances might improve ESAM antibody performance and reliability?

Several technological advances may enhance ESAM antibody performance:

• Recombinant antibody technology: Development of recombinant anti-ESAM antibodies with improved specificity and batch-to-batch consistency

• Nanobodies and single-domain antibodies: Creation of smaller antibody formats for improved tissue penetration and reduced immunogenicity

• Advanced validation methods: Implementation of CRISPR-based validation approaches to confirm antibody specificity

• Multiepitope detection: Development of antibody panels targeting different ESAM epitopes for improved specificity and signal amplification

• Machine learning algorithms: Application of AI-based approaches to optimize antibody selection and experimental conditions

• Antibody engineering: Creation of application-specific anti-ESAM antibodies optimized for particular techniques

These methodological advances promise to enhance the reliability and utility of ESAM antibodies in research applications.

How might ESAM research contribute to understanding disease mechanisms?

ESAM research has significant potential to illuminate disease mechanisms in several areas:

• Vascular disorders: Understanding ESAM's role in regulating vascular permeability may provide insights into conditions characterized by barrier dysfunction

• Hematological disorders: ESAM's contribution to definitive hematopoiesis suggests potential involvement in developmental hematological abnormalities

• Inflammatory diseases: ESAM's function during neutrophil extravasation points to possible roles in inflammatory pathologies

• Cancer biology: As an endothelial junction protein, ESAM may influence tumor angiogenesis and metastasis

• Developmental disorders: ESAM's importance in HSC development suggests potential implications for congenital hematopoietic abnormalities

• Regenerative medicine: Insights from ESAM research may inform approaches to ex vivo HSC expansion and transplantation