ENOSF1 Antibody

What is ENOSF1 and why is it significant in research?

ENOSF1 (Enolase Superfamily Member 1) is a protein belonging to the enolase superfamily with significant implications in both developmental biology and oncology. Research using vertebrate models, particularly zebrafish, has demonstrated that the β splice form (ENOSF1β) plays a crucial role in embryonic development, especially in notochord formation . During early development, ENOSF1β is initially expressed ubiquitously throughout the embryo, with expression becoming restricted to the notochord as development progresses . The protein's significance extends to cancer biology, as clinical studies have implicated ENOSF1β as a risk factor in multiple cancers, including breast and colon cancers . The relationship between ENOSF1 and cancer appears to involve apoptotic regulation, with evidence suggesting that increased ENOSF1β expression may confer a survival advantage to tumor cells by interfering with apoptotic pathways . This dual significance in development and cancer makes ENOSF1 a compelling target for multidisciplinary research.

What are the validated applications for ENOSF1 antibodies in research?

ENOSF1 antibodies have been validated for specific research applications, primarily for protein detection and characterization. The most thoroughly validated applications include:

• Western Blotting (WB): This technique allows detection of ENOSF1 protein in cell or tissue lysates, with recommended dilutions typically ranging from 1:500-2000 . Western blotting is particularly useful for identifying the specific ENOSF1 isoform being expressed, as the calculated molecular weight of approximately 49.8 kDa can be verified .

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of ENOSF1, typically using dilutions of 1:5000-20000 . This approach enables precise quantification of ENOSF1 levels across different samples or experimental conditions.

While not specifically validated for all commercial antibodies, additional potential applications include immunohistochemistry (IHC) and immunofluorescence for localization studies. These techniques are particularly relevant for developmental biology research examining ENOSF1 expression patterns during embryogenesis, as demonstrated in zebrafish studies .

What are the critical specifications to consider when selecting an ENOSF1 antibody?

When selecting an ENOSF1 antibody for research, several critical specifications must be evaluated to ensure experimental success:

• Reactive Species: Confirm that the antibody has been validated for your species of interest. For example, some ENOSF1 antibodies are specifically reactive to human ENOSF1 . If working with model organisms, cross-reactivity must be verified through sequence alignment or preliminary testing.

• Clonality and Host: Most available ENOSF1 antibodies are polyclonal (e.g., rabbit polyclonal) rather than monoclonal . Polyclonal antibodies offer broader epitope recognition but potentially higher background, while monoclonal antibodies provide greater specificity for a single epitope.

• Immunogen Information: Examine which region of ENOSF1 was used as the immunogen. This is particularly important when studying specific splice variants like ENOSF1β . Some antibodies are raised against synthesized peptides derived from specific regions of the human protein .

• Validated Applications: Ensure the antibody has been validated for your intended application (WB, ELISA, IHC, etc.) . Performance can vary significantly between applications, even for the same antibody.

• Storage Conditions: Most ENOSF1 antibodies are supplied in liquid form in PBS containing 50% glycerol and 0.02% sodium azide . Follow manufacturer recommendations for storage temperature (-20°C for long-term, 4°C for frequent use) and avoid repeated freeze-thaw cycles.

The calculated molecular weight of ENOSF1 (49.8 kDa) should be considered when designing experiments to ensure proper identification of the target protein .

How should Western Blot protocols be optimized for reliable ENOSF1 detection?

Optimizing Western Blot protocols for reliable ENOSF1 detection requires careful attention to several methodological factors:

Special considerations for troubleshooting include:

• If signal is weak, increase antibody concentration incrementally (e.g., to 1:500) or extend primary antibody incubation time.

• If background is high, increase blocking time or washing steps, or further dilute primary antibody.

• For multiple isoforms, consider using gradient gels (8-16%) to better resolve potential splice variants.

• Always include positive control lysates if available, particularly from tissues with documented ENOSF1 expression, such as developing notochord in zebrafish models .

• Strip and reprobe membranes with housekeeping protein antibodies (β-actin, GAPDH) to normalize loading and facilitate quantitative analysis.

These optimized protocol parameters create a robust foundation for reliable ENOSF1 detection via Western blotting.

What controls and validation steps are essential when using ENOSF1 antibodies?

Implementation of rigorous controls and validation steps is critical for ensuring reliable results with ENOSF1 antibodies:

• Positive Controls:

  • Cell lines or tissues with confirmed ENOSF1 expression

  • Recombinant ENOSF1 protein (if available)

  • Tissues with documented expression patterns, such as notochord in zebrafish embryos

• Negative Controls:

  • ENOSF1 knockdown samples using validated siRNA or shRNA

  • Secondary antibody-only controls to assess non-specific binding

  • Tissues known to lack ENOSF1 expression

• Specificity Validation:

  • Peptide competition assay: pre-incubate antibody with excess immunogenic peptide, which should eliminate specific signal

  • Multi-antibody approach: use antibodies targeting different ENOSF1 epitopes and compare results

  • Genetic validation: compare antibody signal in wildtype vs. ENOSF1-depleted samples

• Cross-Reactivity Assessment:

  • Test for potential cross-reactivity with other enolase superfamily members

  • If working with non-human samples, verify species cross-reactivity

  • Examine specificity through mass spectrometry analysis of immunoprecipitated bands

• Technical Validation:

  • Verify that detected band matches the expected molecular weight (~49.8 kDa)

  • Establish reproducibility through multiple experimental replicates

  • Compare protein detection with mRNA expression data

These validation steps should be performed before using ENOSF1 antibodies in critical experiments, particularly since ENOSF1 is less well-characterized than many common research targets. Thorough validation ensures that experimental observations truly reflect ENOSF1 biology rather than technical artifacts.

How can ENOSF1 expression be accurately quantified in tissue samples?

Accurate quantification of ENOSF1 expression in tissue samples requires a methodical approach combining multiple techniques:

• Immunohistochemistry (IHC) Quantification:

  • Use automated staining platforms for consistency

  • Develop standardized scoring system (H-score, Allred score, or digital quantification)

  • Include calibration samples in each batch

  • Implement multi-observer scoring to minimize subjective bias

  • Correlate with internal control proteins to normalize across samples

• Western Blot Densitometry:

  • Use standard curves with recombinant protein for absolute quantification

  • Normalize to multiple housekeeping proteins

  • Implement total protein normalization methods (Ponceau S, REVERT stain)

  • Utilize digital image analysis software with linear dynamic range

  • Run technical triplicates to establish reproducibility

• ELISA-Based Quantification:

  • Develop sandwich ELISA using capture and detection antibodies

  • Create standard curves using recombinant ENOSF1

  • Calculate exact protein concentrations in pg/ml or ng/ml

  • Validate assay linearity, precision, and accuracy

  • Test for potential matrix effects from tissue lysates

• Mass Spectrometry Validation:

  • Use targeted mass spectrometry (PRM or SRM) for validation

  • Quantify using isotope-labeled peptide standards

  • Focus on ENOSF1-specific peptides identified through discovery proteomics

  • Correlate with antibody-based quantification methods

• Spatial Analysis:

  • Combine with digital pathology for spatial distribution analysis

  • Quantify expression in specific cell types using multiplex immunofluorescence

  • Correlate with clinical parameters or developmental stages

Particular attention should be paid to the developmental and tissue-specific expression patterns of ENOSF1, such as its restriction to the notochord during zebrafish development . This systematic approach ensures reliable quantification across diverse experimental contexts.

How can ENOSF1 antibodies be used to investigate developmental processes?

ENOSF1 antibodies can be strategically deployed to investigate developmental processes, building on findings from zebrafish studies :

• Spatiotemporal Expression Mapping:

  • Perform whole-mount immunostaining at sequential developmental timepoints

  • Track ENOSF1 expression as it transitions from ubiquitous to notochord-restricted patterns

  • Create high-resolution 3D reconstructions using confocal microscopy

  • Compare protein localization with mRNA expression (from in situ hybridization) to identify post-transcriptional regulation

• Co-localization Analysis:

  • Conduct double immunostaining with ENOSF1 and markers of:

    • Cell proliferation (phospho-histone H3)

    • Apoptosis (cleaved caspase-3)

    • Notochord development (Brachyury/ntl)

    • Cell signaling pathway components (Hedgehog, BMP, TGFβ, FGF)

  • Quantify co-localization to establish functional relationships

• Functional Perturbation Studies:

  • Combine ENOSF1 knockdown (morpholinos) with immunostaining of developmental markers

  • Track changes in notochord structure and integrity

  • Correlate morphological alterations with patterns of apoptosis in peri-notochord tissues

  • Examine effects on pronephros formation and anterior-posterior axis development

• Lineage Tracing Integration:

  • Combine ENOSF1 immunostaining with genetic lineage tracing

  • Track ENOSF1-expressing cells through developmental trajectories

  • Determine if ENOSF1 expression predicts specific cell fates or behaviors

• Cross-Species Comparative Studies:

  • Apply validated antibodies across diverse vertebrate models

  • Map evolutionary conservation of ENOSF1 expression patterns

  • Correlate expression with morphological innovations in the notochord

This integrated approach can elucidate ENOSF1's role in vertebrate development, particularly in notochord formation and the regulation of apoptosis during embryogenesis, extending our understanding beyond the initial zebrafish findings .

What insights have been gained about ENOSF1's role in notochord development?

Research on ENOSF1, particularly using zebrafish models, has provided several key insights into its role in notochord development:

• Expression Pattern Dynamics:

  • ENOSF1β (enosf1b) is initially expressed ubiquitously throughout early embryos

  • Expression becomes progressively restricted to the notochord during somitogenesis

  • By 48-72 hours post-fertilization (hpf), expression is predominantly in the notochord

  • At 96 hpf, notochord expression diminishes with residual expression in the pancreas

• Functional Requirement:

  • Morpholino-mediated knockdown of enosf1b results in kinked notochords

  • Embryos display shortened anterior-posterior axes

  • Circulatory edema develops in knockdown embryos

  • These phenotypes indicate an essential role in notochord development and function

• Cell Survival Regulation:

  • ENOSF1β knockdown increases apoptosis specifically in peri-notochord tissues

  • This occurs independently of cell cycle arrest, as demonstrated by phospho-histone H3 staining

  • The effect is p53-independent, suggesting a direct role in survival signaling

• Signaling Hypothesis:

  • ENOSF1β may participate in producing pro-survival signals from the notochord

  • Alternatively, it might be involved in consuming or processing pro-death signals

  • This signaling role aligns with the known function of the notochord as a signaling center

• Broader Developmental Impact:

  • Notochord defects in ENOSF1β-depleted embryos affect adjacent tissues

  • Whole mount in situ hybridization for ntl or pax2a shows deformed notochord and pronephros

  • These findings suggest ENOSF1β's influence extends beyond the notochord itself

These insights position ENOSF1 as an important regulator of vertebrate embryonic development, particularly in the formation and signaling functions of the notochord. The connection to apoptosis regulation has significant implications for understanding its potential role in cancer biology as well .

How does ENOSF1 expression change during embryonic development?

ENOSF1 expression undergoes distinct temporal and spatial changes during embryonic development, as revealed by zebrafish studies:

The dynamic regulation of ENOSF1 during development suggests several important mechanisms:

• Transcriptional Regulation: The progressive restriction from ubiquitous to tissue-specific expression indicates developmental stage-specific transcriptional control.

• Tissue-Specific Functions: The persistence in notochord suggests specialized functions in this tissue, potentially related to structural integrity or signaling.

• Functional Transitions: The shift from notochord to pancreas expression at later stages suggests evolving roles during organogenesis.

• Post-Transcriptional Control: RT-PCR analysis confirmed ENOSF1 expression throughout early embryonic development, with whole mount in situ hybridization revealing the spatial restrictions .

This expression pattern correlates with phenotypic effects observed in knockdown studies, where notochord defects and increased peri-notochord apoptosis were prominent . The precise timing of expression changes aligns with critical developmental events, particularly notochord formation and function, supporting ENOSF1's role as a developmental regulator.

What is the relationship between ENOSF1 and cancer progression?

The relationship between ENOSF1 and cancer progression is emerging as an important area of research, with several key findings:

• Clinical Associations:

  • ENOSF1β has been implicated as a risk factor in multiple human cancers

  • Studies have identified associations with breast and colon cancers

  • ENOSF1 polymorphisms have been linked to treatment response in gastric cancer patients

• Apoptosis Regulation:

  • Increased apoptosis following ENOSF1β knockdown in zebrafish suggests a pro-survival function

  • This implies that increased ENOSF1β expression might confer a survival advantage to tumor cells

  • The mechanism appears to be independent of cell cycle regulation, as demonstrated by phospho-histone H3 staining

• Treatment Response Correlation:

  • ENOSF1 polymorphisms (rs2612091, rs2741171) have been studied in relation to 5-fluorouracil (5FU) treatment response in gastric cancer

  • These genetic variations may affect how patients respond to chemotherapy

  • The connection suggests ENOSF1 may influence drug metabolism or cellular survival pathways

• Potential Mechanisms:

  • ENOSF1β may participate in producing soluble survival signals, similar to observations in drug-resistant cancer cells

  • It could function in metabolic pathways that support cancer cell survival

  • Its developmental role suggests potential involvement in dedifferentiation processes in cancer

• Therapeutic Implications:

  • The increased apoptosis following ENOSF1 knockdown suggests it could be a therapeutic target

  • Targeting ENOSF1 catalytic activity or protein-protein interactions might increase apoptosis in tumors

  • ENOSF1 status might serve as a biomarker for treatment selection, particularly for 5FU-based therapies

These findings collectively suggest that ENOSF1 may contribute to cancer progression by enhancing cell survival and potentially modulating response to chemotherapy. The dual implications in both development and cancer highlight ENOSF1 as a molecular target worthy of further investigation in oncology research.

How can ENOSF1 polymorphisms be studied in relation to chemotherapy response?

Studying ENOSF1 polymorphisms in relation to chemotherapy response requires a methodical approach combining genetic analysis with protein expression and functional studies:

This comprehensive approach enables researchers to establish causal relationships between ENOSF1 polymorphisms and treatment response, potentially leading to personalized treatment strategies for cancer patients.

What techniques can be used to investigate ENOSF1's role in cancer cell survival?

Investigating ENOSF1's role in cancer cell survival requires a multifaceted approach combining molecular, cellular, and functional techniques:

• Expression Modulation Techniques:

  • RNA interference (siRNA/shRNA) for transient or stable ENOSF1 knockdown

  • CRISPR-Cas9 gene editing for complete knockout or mutation introduction

  • Lentiviral overexpression systems for gain-of-function studies

  • Inducible expression systems to study temporal requirements

• Apoptosis and Cell Survival Assays:

  • Annexin V/PI staining followed by flow cytometry to quantify apoptotic cell populations

  • TUNEL assay to detect DNA fragmentation in apoptotic cells

  • Caspase activity assays (caspase-3/7, -8, -9) to determine apoptotic pathway activation

  • Mitochondrial membrane potential assays to assess intrinsic apoptosis pathway

  • Colony formation assays to evaluate long-term survival capacity

• Mechanism Investigation:

  • Co-immunoprecipitation with ENOSF1 antibodies to identify binding partners

  • Western blotting to assess activation status of survival pathways (Akt, ERK, NF-κB)

  • Phospho-protein arrays to broadly screen pathway activation

  • Proximity ligation assay (PLA) to detect protein-protein interactions in situ

  • Subcellular fractionation to determine ENOSF1 localization during apoptotic stress

• Contextual Influences:

  • 3D culture systems (spheroids, organoids) to assess survival in physiologically relevant contexts

  • Co-culture systems to examine paracrine effects on surrounding cells

  • Drug response profiling across multiple cancer therapeutics

  • Nutrient and oxygen deprivation studies to mimic tumor microenvironment stress

• In Vivo Validation:

  • Xenograft models with ENOSF1-modulated cancer cells

  • Patient-derived xenografts (PDX) with characterized ENOSF1 status

  • Analysis of tumor growth, metastasis, and therapy response

  • Immunohistochemistry with ENOSF1 antibodies to assess expression in tumor sections

These methodologies can systematically dissect ENOSF1's role in cancer cell survival, building on the observations from developmental studies that demonstrated increased apoptosis following ENOSF1 knockdown . Understanding these mechanisms could potentially lead to novel therapeutic strategies targeting ENOSF1 in cancer.

What are the challenges in detecting specific ENOSF1 isoforms and how can they be overcome?

Detecting specific ENOSF1 isoforms presents several technical challenges that require specialized approaches:

• Challenge: Multiple Splice Variants

  • Solution: Isoform-Specific Epitope Targeting

    • Verify antibody epitope location relative to known splice junctions

    • Use multiple antibodies targeting different protein regions

    • Develop custom antibodies against unique splice junctions

    • Complement with RT-PCR using splice variant-specific primers as demonstrated in zebrafish studies

• Challenge: Similar Molecular Weights

  • Solution: High-Resolution Separation Techniques

    • Employ gradient gels (8-16%) for superior resolution

    • Use extended SDS-PAGE running times

    • Consider Phos-tag™ gels if phosphorylation differentiates isoforms

    • Implement 2D gel electrophoresis (isoelectric focusing followed by SDS-PAGE)

    • Verify identity through mass spectrometry of excised bands

• Challenge: Cross-Reactivity with Related Proteins

  • Solution: Enhanced Specificity Validation

    • Perform extensive blocking peptide competition assays

    • Include genetic controls (knockdown/knockout of specific isoforms)

    • Use recombinant protein standards representing each isoform

    • Apply orthogonal detection methods like targeted mass spectrometry

    • Validate results across multiple antibody clones

• Challenge: Differential Subcellular Localization

  • Solution: Spatial Resolution Techniques

    • Conduct subcellular fractionation before Western blotting

    • Employ high-resolution confocal microscopy with co-localization markers

    • Implement super-resolution microscopy (STORM, STED) for nanoscale localization

    • Use proximity ligation assay to detect isoform-specific interactions

• Challenge: Low Abundance of Specific Isoforms

  • Solution: Signal Enhancement Methods

    • Implement immunoprecipitation before Western blotting

    • Use tyramide signal amplification for immunohistochemistry

    • Apply nested PCR approaches for transcript detection

    • Consider targeted proteomics (PRM/SRM) for sensitive detection

These approaches can be particularly valuable when studying the β splice form of ENOSF1 (ENOSF1β), which has been implicated in developmental processes and cancer biology . Careful attention to isoform-specific detection enables researchers to distinguish the potentially distinct functions of different ENOSF1 variants.

How can protein-protein interactions of ENOSF1 be effectively studied?

Investigating ENOSF1 protein-protein interactions requires a strategic combination of complementary techniques:

• Antibody-Based Interaction Discovery:

  • Co-Immunoprecipitation (Co-IP)

    • Use validated ENOSF1 antibodies to pull down protein complexes

    • Analyze co-precipitated proteins by mass spectrometry

    • Confirm interactions by reverse Co-IP with antibodies against identified partners

    • Compare interactome across different cellular contexts or developmental stages

• In Situ Interaction Validation:

  • Proximity Ligation Assay (PLA)

    • Combine ENOSF1 antibody with antibodies against putative interaction partners

    • Visualize interactions as fluorescent spots in fixed cells or tissues

    • Quantify interaction frequency in different subcellular compartments

    • Examine how interactions change during development or disease progression

• Live-Cell Interaction Dynamics:

  • Fluorescence Resonance Energy Transfer (FRET)

    • Generate fluorescent protein-tagged ENOSF1 constructs

    • Measure energy transfer between ENOSF1 and potential partners

    • Track dynamic interactions in real-time in living cells

    • Analyze how interactions respond to cellular stimuli or stress conditions

• Domain-Specific Interaction Mapping:

  • Protein Fragment Complementation Assays

    • Create domain-specific constructs to map interaction interfaces

    • Use techniques like split-luciferase or split-GFP complementation

    • Determine which regions of ENOSF1 mediate specific interactions

    • Generate interaction-deficient mutants for functional studies

• High-Throughput Screening Approaches:

  • Yeast Two-Hybrid or Mammalian Two-Hybrid

    • Screen for novel ENOSF1 interactors from cDNA libraries

    • Validate hits using orthogonal methods

    • Integrate with existing protein interaction networks

    • Focus on developmental or cancer-related libraries based on ENOSF1's dual roles

• Structural Interaction Analysis:

  • Cross-linking Mass Spectrometry (XL-MS)

    • Stabilize transient interactions with chemical cross-linkers

    • Identify interacting regions with amino acid resolution

    • Map interaction interfaces onto protein structural models

    • Guide the development of interaction-disrupting compounds

This systematic approach can reveal ENOSF1's functional partners, providing insights into its mechanisms in both developmental processes and cancer pathways. Given ENOSF1's potential role in producing or regulating survival signals , mapping its interaction network is particularly valuable for understanding its biological functions.

What are best practices for quantitative analysis of ENOSF1 expression across different tissue samples?

Implementing best practices for quantitative analysis of ENOSF1 expression across tissue samples requires rigorous methodology:

• Sample Collection and Processing Standardization:

  • Establish consistent tissue collection protocols

  • Standardize fixation parameters (type, duration, temperature)

  • Create detailed SOPs for tissue processing and storage

  • Document preservation method and storage duration for each sample

  • Consider tissue microarrays for large-scale studies to minimize batch effects

• Multi-Platform Quantification Approach:

  • Western Blot Quantification

    • Use validated ENOSF1 antibodies at optimized dilutions

    • Include recombinant protein standards for absolute quantification

    • Apply total protein normalization (Ponceau S, REVERT) rather than single housekeeping genes

    • Employ digital image analysis with linear dynamic range

    • Analyze technical triplicates to establish reproducibility

  • Immunohistochemistry Quantification

    • Implement automated staining platforms for consistency

    • Develop standardized scoring system (H-score, Allred, or digital analysis)

    • Conduct multi-observer scoring to minimize subjective bias

    • Integrate machine learning algorithms for unbiased quantification

    • Use multispectral imaging to differentiate specific from non-specific staining

• Statistical Analysis Framework:

  • Power analysis to determine appropriate sample size

  • Implement hierarchical linear models for nested data structures

  • Account for batch effects through appropriate statistical corrections

  • Utilize biostatistician input for complex experimental designs

  • Apply appropriate multiple testing corrections for large-scale studies

• Quality Control Measures:

  • Include both positive and negative control tissues in each batch

  • Incorporate spike-in standards where applicable

  • Validate a subset of samples using orthogonal methods

  • Establish acceptance criteria for assay variability

  • Implement blinding procedures for analysis

• Data Integration and Reporting:

  • Correlate protein expression with genetic data (e.g., ENOSF1 polymorphisms)

  • Integrate with relevant clinical or developmental parameters

  • Follow standardized reporting guidelines (REMARK for biomarker studies)

  • Deposit raw data in appropriate repositories for transparency

  • Document all antibody information (catalog number, lot, dilution)

This comprehensive approach ensures reliable quantification of ENOSF1 across diverse tissue samples, allowing for meaningful comparisons in both developmental biology and cancer research contexts.