EPAS1 Antibody, FITC conjugated

What is EPAS1 and why is it significant in biological research?

EPAS1 (endothelial PAS domain protein 1) is a hypoxia-inducible transcription factor that plays a critical role in the body's adaptation to changing oxygen levels. It functions as the alpha subunit of a larger protein complex called HIF (Hypoxia-Inducible Factor), which regulates genes involved in cell division, angiogenesis, and erythropoiesis. EPAS1 is particularly significant because it serves as a major regulator of erythropoietin, controlling red blood cell production .

In research contexts, EPAS1 has emerged as a fundamental molecule for studying hypoxic responses in various tissues. Recent studies have demonstrated its essential role in spermatogonial stem cell (SSC) function, where it regulates key cellular processes such as metabolism in regenerative conditions . Additionally, EPAS1 contributes to the maintenance of primordial follicles in female reproductive systems and has significant implications in cardiovascular research .

What distinguishes the FITC-conjugated EPAS1 antibody from other detection methods?

FITC (Fluorescein Isothiocyanate) conjugation provides direct fluorescent visualization of EPAS1 without requiring secondary antibody steps, streamlining immunofluorescence workflows. The commercially available FITC-conjugated EPAS1 antibody is a polyclonal antibody developed in rabbits using recombinant human EPAS1 protein (amino acids 543-870) as the immunogen .

This conjugation offers several advantages over unconjugated antibodies, including:

• Direct visualization without secondary antibody incubation steps

• Reduced background from non-specific secondary antibody binding

• Compatibility with multicolor immunofluorescence when combined with antibodies conjugated to spectrally distinct fluorophores

• Quantitative analysis through fluorescence intensity measurements

While the commercially available FITC-conjugated EPAS1 antibody has been validated for ELISA applications , researchers should conduct validation for other applications such as immunocytochemistry, flow cytometry, or immunohistochemistry.

How is EPAS1 regulated physiologically, and how does this affect experimental design?

EPAS1 regulation is primarily oxygen-dependent through a sophisticated post-translational mechanism. Under normoxic conditions (adequate oxygen), EPAS1/HIF-2α is continuously produced but rapidly targeted for degradation by oxygen-sensing proteins . During hypoxia, EPAS1 degradation slows significantly, allowing it to accumulate and form functional transcription complexes that activate hypoxia-responsive genes .

This oxygen-dependent regulation has critical implications for experimental design:

• Cell culture oxygen conditions must be precisely controlled and reported

• Sample collection and processing should minimize oxygen fluctuations

• Time between tissue harvesting and fixation/analysis should be minimized

• Positive controls under known hypoxic conditions should be included

• Researchers may need to consider using prolyl hydroxylase inhibitors like Daprodustat when studying EPAS1 in vitro, as shown in recent studies where Daprodustat treatment resulted in a "consistent and significant dose-dependent increase in EPAS1 expression"

The dynamic nature of EPAS1 expression means that experimental timing is crucial, as demonstrated in studies showing significant changes in EPAS1-positive cell populations within just 24 hours of treatment .

What are the optimal protocols for using FITC-conjugated EPAS1 antibody in immunofluorescence studies?

For optimal immunofluorescence results with FITC-conjugated EPAS1 antibody, the following protocol is recommended based on research applications:

Sample Preparation:

• Fix cells/tissues with 4% paraformaldehyde for 15-20 minutes at room temperature

• Permeabilize with 0.1-0.5% Triton X-100 for 10 minutes

• Block with 5-10% normal serum (matching secondary host if using unconjugated antibodies) with 1% BSA for 1 hour

Antibody Incubation:

• Dilute FITC-conjugated EPAS1 antibody to 1:100-1:500 in blocking buffer

• Incubate overnight at 4°C in a humidified chamber

• Wash 3-5 times with PBS containing 0.05% Tween-20

Counterstaining and Mounting:

• Add nuclear counterstain (e.g., DAPI at 1:1000) for 5-10 minutes

• Mount with anti-fade mounting medium

• Seal with nail polish and store at 4°C protected from light

Critical Considerations:

• When studying spermatogonial stem cells, coordinate with ID4-EGFP labeling for proper identification, as studies show 95-100% of SSCs are EPAS1-positive compared to only 4.3-6.5% of progenitor cells

• Include appropriate controls including negative controls (secondary antibody only) and positive controls (tissues known to express EPAS1, such as vascularized tissues)

• Optimize antibody concentration for your specific tissue/application, as expression levels vary significantly between cell types

How can EPAS1 antibody be used to assess hypoxic conditions in tissue samples?

EPAS1 antibody can be effectively combined with hypoxia markers to assess tissue oxygen status through a dual-labeling approach:

Protocol for Hypoxia Assessment:

• Inject experimental animals with pimonidazole (Hypoxyprobe, 60mg/kg IP) 90-120 minutes before tissue collection

• Process tissues quickly and fix in 4% paraformaldehyde

• Section tissues at 5-7μm thickness

• Perform dual immunostaining with:

  • Anti-pimonidazole antibody to detect hypoxic regions

  • FITC-conjugated EPAS1 antibody to visualize EPAS1 expression

Analysis Considerations:

• Quantify co-localization of pimonidazole adducts and EPAS1 expression

• Assess protein expression via immunoblotting as a complementary approach

• Compare EPAS1 expression between different cell populations using FACS if appropriate

Recent studies demonstrate this approach's effectiveness, showing that over 80% of spermatogonial stem cells reside in hypoxic conditions in the testis, with significantly higher levels of pimonidazole adducts identified in SSCs compared to progenitor spermatogonia . Immunoblotting analysis normalized to tubulin revealed that pimonidazole adducts were particularly prominent in proteins of molecular weight 50-170 kDa in SSC lysates .

What controls should be included when using EPAS1 antibody for Western blotting?

When performing Western blot analysis with EPAS1 antibody, comprehensive controls are essential for reliable interpretation:

Essential Controls:

Technical Considerations:

• Use a 5-20% SDS-PAGE gel for optimal resolution of the EPAS1 protein (120 kDa)

• Transfer conditions should be optimized for large proteins (e.g., 150 mA for 50-90 minutes)

• Block with 5% milk for 2 hours at room temperature

• Dilute primary EPAS1 antibody to 1:1000 in 5% milk and incubate at 4°C overnight with rotation

• Wash thoroughly with TBS-0.1% Tween (3 × 5 minutes)

• Use high-sensitivity chemiluminescent substrate for detection

Published research shows that a specific band for EPAS1 should be detected at approximately 120 kDa, which aligns with the expected molecular weight .

How should experiments be designed to study EPAS1 in stem cell populations?

Designing experiments to study EPAS1 in stem cell populations requires careful consideration of several factors:

Recommended Experimental Design:

• Cell Isolation Strategy

  • For spermatogonial stem cells: Utilize ID4-EGFP transgenic mouse models for fluorescence-based identification

  • Implement FACS with appropriate gating parameters to separate stem cell populations (e.g., ID4-EGFP Bright) from progenitor populations (ID4-EGFP Dim)

• Culture Conditions Management

  • Monitor and control oxygen levels precisely (standard culture conditions often use 5% CO2, 10% O2)

  • Consider using prolyl hydroxylase inhibitors like Daprodustat to prevent EPAS1 degradation in vitro

  • Document passage number, as EPAS1 expression significantly decreases with extended culture (5-10 passages)

• Functional Assessment Approaches

  • Transplantation assays to assess stem cell regenerative capacity

  • Molecular profiling through RNA-sequencing to identify EPAS1-regulated genes

  • Metabolic assays to evaluate EPAS1's impact on cellular energetics

Research demonstrates that EPAS1 manipulation significantly affects stem cell function. For example, Daprodustat treatment increased the percentage of ID4-EGFP+ cells and the mean fluorescence intensity of EGFP after just 24 hours . More importantly, transplantation analyses confirmed a significant 3-fold increase in SSC-derived colonies following transplantation of Daprodustat-treated spermatogonia versus controls .

What are the methodological considerations when studying EPAS1 in reproductive biology?

EPAS1 research in reproductive biology requires specialized approaches for both male and female systems:

Male Reproductive System Methodology:

• Use transgenic models (e.g., Id4-eGfp) to visualize spermatogonial stem cells

• Consider conditional knockout approaches using germline-specific Cre recombinase systems (Ddx4-cre)

• Employ pimonidazole labeling for in situ hypoxia assessment

• Evaluate stem cell function through:

  • Post-transplantation regeneration assays

  • Post-chemotherapy recovery models

  • Sperm output, motility, and viability measurements

Female Reproductive System Methodology:

• Focus assessment on primordial follicles where EPAS1 expression is prominent

• Be cautious with antibody specificity, as non-specific zona pellucida cross-reactivity has been reported in immunostaining

• Differentiate between germline-specific EPAS1 expression and somatic cell expression throughout the ovarian cortex and within granulosa cells

Common Technical Considerations:

• Implement Cre/loxP recombination technology for targeted knockouts to distinguish between germline-specific and global EPAS1 functions

• Design experimental controls that account for the expression of EPAS1 in both germ cells and supporting somatic cells

• Document developmental stage precisely, as EPAS1 expression patterns change during reproductive system development

Research demonstrates that EPAS1 is required for robust SSC function specifically under regenerative conditions, while showing more modest impacts under steady-state conditions . Similarly, in female reproduction, EPAS1 ablation results in significant fertility reduction, though less severe than in global knockout models .

How can RNA-sequencing data be used to identify EPAS1-regulated pathways?

RNA-sequencing provides powerful insights into EPAS1-regulated pathways when correctly designed and analyzed:

Experimental Design for RNA-seq:

• Compare control samples with those where EPAS1 activity is modulated through:

  • Pharmacological intervention (e.g., Daprodustat treatment)

  • Genetic manipulation (conditional knockout models)

  • Hypoxia/normoxia comparison

• Include adequate biological replicates (minimum n=4 per group)

• Process samples for bulk RNA-seq or single-cell RNA-seq depending on research question

Analytical Pipeline:

• Impose appropriate statistical thresholds (e.g., false discovery rate <0.05)

• Categorize differentially expressed genes (upregulated/downregulated with appropriate fold-change thresholds)

• Perform Gene Ontology (GO) analysis to identify enriched pathways

• Validate key findings through complementary approaches:

  • RT-qPCR for selected genes

  • Protein expression analysis

  • Functional assays specific to identified pathways

Research Example:
A bulk RNA-seq comparison of control and Daprodustat-treated undifferentiated spermatogonia (n=4) revealed 3,604 differentially expressed genes (FDR<0.05), with 1,759 upregulated following treatment (582 with fold change ≥2) and 1,845 downregulated (276 with fold change ≤-2) . Gene Ontology analysis of these differentially expressed genes provided insights into the key cellular processes controlled by EPAS1-regulated genes, highlighting metabolic pathways as particularly significant targets .

How can researchers resolve common issues with EPAS1 antibody staining?

When working with FITC-conjugated EPAS1 antibody, several challenges may arise. Here are methodological solutions for common problems:

Problem: High Background Fluorescence

• Solution: Implement additional blocking steps using 5-10% normal serum from the host species of the primary antibody, combined with 1% BSA

• Method: Pre-incubate sections in blocking solution for 1-2 hours at room temperature before antibody addition

• Alternative: Include 0.1-0.3% Triton X-100 in blocking solution to reduce non-specific membrane binding

Problem: Weak or Absent EPAS1 Signal

• Solution: Optimize antigen retrieval methods

• Method: Test heat-mediated retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) at 95-98°C for 15-20 minutes

• Alternative: Increase antibody concentration or incubation time, and ensure samples were properly fixed

Problem: Non-specific Zona Pellucida Staining

• Solution: Implement more stringent controls

• Method: Include samples from Epas1-cKO animals as negative controls, as research shows persistent zona pellucida cross-reactivity even in knockout tissues

• Alternative: Use multiple EPAS1 antibodies targeting different epitopes to confirm specificity

Problem: Inconsistent EPAS1 Detection Across Experiments

• Solution: Standardize oxygen conditions during sample processing

• Method: Process all samples rapidly and consistently, minimizing time between collection and fixation

• Alternative: Consider using in vivo hypoxia markers like pimonidazole administered before tissue collection

How should researchers interpret variations in EPAS1 expression between different cell populations?

Interpreting EPAS1 expression patterns requires careful consideration of multiple factors:

Methodological Approach to Interpretation:

• Quantitative Assessment Methods

  • Use densitometry analysis normalized to housekeeping proteins (e.g., tubulin) for Western blots

  • Determine percentage of EPAS1+ cells within specific populations using co-staining approaches

  • Measure fluorescence intensity as a proxy for expression level

• Cell Type-Specific Benchmarks

  • Compare to known expression patterns: In testes, 95-100% of SSCs express EPAS1 versus only 4.3-6.5% of progenitor cells

  • Consider developmental stage: EPAS1 expression in ovarian tissue varies between P4 and P14 developmental stages

  • Examine tissue vascularization: EPAS1 is expressed at higher levels in highly vascularized tissues like placenta and lung

• Biological Context Integration

  • Correlate EPAS1 expression with functional outcomes (e.g., stem cell activity, regenerative capacity)

  • Assess relationship to hypoxia markers like pimonidazole adducts

  • Consider relationship to disease states (e.g., familial erythrocytosis associated with EPAS1 mutations)

When analyzing data, it's important to note that even modest differences in EPAS1 expression can have significant biological impact. For example, a 50% reduction in EPAS1 expression in progenitor spermatogonia compared to SSCs corresponds with substantial differences in stem cell function .

What are the key considerations when interpreting experiments using hypoxia-inducing agents versus genetic manipulation of EPAS1?

Researchers must understand the distinct implications of different EPAS1 manipulation approaches:

Comparative Analysis Framework:

Interpretive Guidelines:

How can EPAS1 antibodies be used in multi-parameter flow cytometry for stem cell research?

Multi-parameter flow cytometry with EPAS1 detection enables sophisticated stem cell population analysis:

Optimized Protocol for Flow Cytometry:

• Sample Preparation

  • Prepare single-cell suspensions from tissues of interest

  • Fix cells with 2-4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1% Triton X-100 or commercial permeabilization buffer

• Staining Strategy

  • Block with 2% BSA in PBS for 30 minutes

  • For EPAS1 detection: Use FITC-conjugated EPAS1 antibody at 1:100-1:200 dilution

  • For stem cell markers: Include appropriate conjugated antibodies (e.g., PE-conjugated)

  • For DNA content: Add far-red DNA dye (7-AAD or DRAQ7)

• Gating Strategy

  • Exclude doublets based on FSC-H vs. FSC-A

  • Remove dead cells using viability dye

  • Identify stem cell populations using established markers

  • Analyze EPAS1 expression within defined populations

Advanced Applications:

• Combine with cell cycle analysis to determine if EPAS1 expression varies with cell cycle stage

• Sort EPAS1-high vs. EPAS1-low populations for downstream functional assays or transcriptomic analysis

• Implement intracellular metabolic dyes to correlate EPAS1 expression with metabolic status

This approach has been validated in research where FACS with specific gating parameters was used to isolate ID4-EGFP Bright (SSC) and ID4-EGFP Dim (progenitor) populations for subsequent EPAS1 analysis . This technique allowed researchers to quantitatively demonstrate significantly higher EPAS1 expression in stem cell populations compared to progenitor cells .

What are the methodological approaches for investigating EPAS1 in metabolic regulation of stem cells?

EPAS1's role in metabolic regulation can be investigated through multiple complementary approaches:

Comprehensive Metabolic Assessment Strategy:

• Transcriptomic Analysis

  • Perform RNA-seq on control versus EPAS1-modulated cells

  • Focus analysis on metabolic pathway genes

  • Validate key metabolic gene expression changes through RT-qPCR

• Metabolic Flux Analysis

  • Measure oxygen consumption rate (OCR) using Seahorse XF analyzer

  • Assess extracellular acidification rate (ECAR) to evaluate glycolytic activity

  • Perform substrate utilization tests with glucose, glutamine, and fatty acid restrictions

• Metabolomic Profiling

  • Conduct targeted metabolomics focusing on glycolytic intermediates, TCA cycle components, and lipid metabolites

  • Compare metabolite profiles between control and EPAS1-modulated cells

  • Trace isotope-labeled substrate metabolism to determine pathway activities

• Functional Enzyme Assays

  • Measure activities of key metabolic enzymes regulated by EPAS1

  • Assess mitochondrial function through membrane potential measurements

  • Evaluate ROS production as an indicator of metabolic stress

Recent research highlights the importance of such approaches, demonstrating that EPAS1 regulates key cellular processes including metabolism in spermatogonial stem cells under regenerative conditions . The application of bulk RNA-sequencing to EPAS1-modulated cells (Daprodustat treatment) revealed significant alterations in gene expression patterns related to metabolic pathways, providing insights into how EPAS1 controls stem cell energetics .

How can researchers design experiments to investigate EPAS1's role in adaptation to environmental stressors?

Investigating EPAS1's role in adaptation to environmental stressors requires specialized experimental designs:

Experimental Framework:

• Altitude Adaptation Studies

  • Compare EPAS1 expression and activity between populations adapted to different altitudes

  • Measure physiological parameters (hematocrit, hemoglobin, oxygen saturation)

  • Assess EPAS1 genetic variants associated with high-altitude adaptation

• Temperature Variation Response

  • Expose cells/organisms to controlled temperature variations

  • Monitor EPAS1 expression, stability, and downstream target activation

  • Evaluate tissue-specific responses in metabolically active versus less active tissues

• Oxidative Stress Models

  • Induce oxidative stress through H2O2 treatment or metabolic inhibitors

  • Measure EPAS1 stabilization and nuclear localization

  • Assess protective effects of EPAS1 activation versus genetic ablation

• Methodology for Tissue-Specific Effects

  • Implement tissue-specific conditional knockout models for EPAS1

  • Compare phenotypes across multiple tissue types

  • Evaluate systemic versus local responses to stressors

Recommended Approach for Adaptation Studies:

• Use multiple timepoints to distinguish between acute and chronic responses

• Combine in vivo and in vitro approaches for comprehensive understanding

• Implement genetic models with human EPAS1 variants to investigate population-specific adaptations

This research direction is particularly relevant given EPAS1's established role in high-altitude adaptation. At higher altitudes, air pressure is lower and less oxygen enters the body, which triggers compensatory mechanisms including changes in breathing patterns and increased production of red blood cells and blood vessels . EPAS1 variants have been identified that influence the relative contribution of aerobic and anaerobic metabolism, affecting maximum sustainable metabolic power for different event durations .

What methodologies are appropriate for studying EPAS1 in the context of reproductive biology disorders?

Investigating EPAS1 in reproductive disorders requires specialized approaches:

Methodological Framework:

• Clinical Sample Analysis

  • Obtain testicular or ovarian biopsies from patients with reproductive disorders

  • Perform immunohistochemistry for EPAS1 using validated antibodies

  • Compare expression patterns with matched controls

• Model Systems Approach

  • Develop reproductive disorder models through:

    • Conditional knockout of EPAS1 in specific cell types using Ddx4-cre (germline) or other tissue-specific Cre drivers

    • Environmental manipulations that affect oxygen tension in reproductive tissues

    • Exposure to reproductive toxicants with monitoring of EPAS1 response

• Functional Recovery Assessment

  • Test interventions targeting EPAS1 (e.g., Daprodustat) in reproductive disorder models

  • Evaluate recovery metrics including:

    • Spermatogonial stem cell repopulation post-chemotherapy

    • Primordial follicle maintenance and development

    • Fertility outcomes and reproductive capacity

Research indicates significant potential in this area, as EPAS1 has been shown to be required for robust SSC function during regeneration after chemotherapy treatment . Similarly, female Epas1 conditional knockout models show significant reduction in fertility, though less severe than global knockouts, suggesting potential therapeutic avenues .

How should researchers design experiments to identify novel EPAS1 interaction partners in stem cell biology?

Identifying EPAS1 protein interactions requires specialized experimental approaches:

Comprehensive Interaction Discovery Strategy:

• Affinity Purification-Mass Spectrometry (AP-MS)

  • Express tagged EPAS1 in relevant stem cell populations

  • Perform immunoprecipitation under both normoxic and hypoxic conditions

  • Analyze by mass spectrometry to identify co-precipitating proteins

  • Validate with reciprocal co-IP experiments

• Proximity Labeling Approaches

  • Generate BioID or APEX2 fusion constructs with EPAS1

  • Express in stem cells under controlled oxygen conditions

  • Identify biotinylated proteins in proximity to EPAS1

  • Compare interaction networks between different cell states

• Yeast Two-Hybrid Screening

  • Use EPAS1 domains as bait against stem cell cDNA libraries

  • Focus on specific domains (PAS domain, transactivation domains)

  • Validate interactions in mammalian cells

• Co-localization Studies

  • Perform dual immunofluorescence with FITC-conjugated EPAS1 antibody and antibodies against candidate interactors

  • Quantify co-localization using confocal microscopy

  • Implement FRET or PLA (Proximity Ligation Assay) for direct interaction evidence

Data Analysis Strategy:

• Compare interaction networks under different oxygen tensions

• Identify stem cell-specific versus common interaction partners

• Integrate with transcriptomic data to correlate physical interactions with gene regulation

This approach is particularly relevant given the complex regulation of EPAS1, which includes interactions with proteins that target it for degradation under normoxic conditions but allow its stabilization during hypoxia .

What experimental designs are most effective for translating EPAS1 research findings into therapeutic applications?

Translating EPAS1 research toward therapeutic development requires strategic experimental designs:

Translational Research Framework:

• Target Validation Strategy

  • Confirm EPAS1's role in specific disease contexts through:

    • Human genetic association studies (e.g., EPAS1 mutations in familial erythrocytosis)

    • Tissue-specific conditional knockout phenotyping

    • Patient sample analysis correlating EPAS1 expression with disease severity

• Therapeutic Modulation Approaches

  • For EPAS1 activation in regenerative conditions:

    • Test prolyl hydroxylase inhibitors like Daprodustat with dose optimization

    • Evaluate delivery systems for tissue-specific targeting

    • Determine treatment windows for maximal efficacy

  • For EPAS1 inhibition in pathological conditions:

    • Develop selective EPAS1 inhibitors that spare HIF1A

    • Test RNA interference approaches for specificity

    • Evaluate downstream target inhibition as alternative strategy

• Preclinical Efficacy Models

  • For male fertility applications:

    • Use chemotherapy-induced testicular damage models

    • Evaluate stem cell transplantation combined with EPAS1 modulation

    • Assess sperm production, motility, and fertility outcomes

  • For female fertility applications:

    • Develop models of premature ovarian insufficiency

    • Evaluate primordial follicle preservation strategies

    • Monitor follicle development and ovulation after treatment

• Safety Assessment Design

  • Monitor for potential off-target effects on erythropoiesis

  • Assess cardiovascular impacts given EPAS1's role in vascular biology

  • Determine optimal therapeutic window between efficacy and safety

The potential for therapeutic application is supported by research showing that Daprodustat treatment significantly improved SSC maintenance in vitro and increased SSC-derived colony formation 3-fold in transplantation studies , suggesting viable pathways to clinical translation for reproductive medicine.