EPAS1 Antibody, HRP conjugated

What is EPAS1 and why is it important in hypoxia research?

EPAS1 (Endothelial PAS domain-containing protein 1), also known as HIF-2α, is a critical transcription factor that regulates cellular responses to hypoxia. It forms heterodimers with HIF-1β (Arnt 1) to bind to hypoxia-responsive elements in target gene promoters, thereby enabling the transcription of genes that help cells adapt to oxygen deprivation. EPAS1 plays crucial roles in angiogenesis, erythropoiesis, and glucose metabolism, making it an essential factor in understanding cellular adaptation to low oxygen conditions . Dysregulation of EPAS1 has been implicated in various pathologies including cancer and cardiovascular diseases, highlighting its importance as a research target .

What is the difference between HIF-1α and EPAS1/HIF-2α in experimental settings?

While both HIF-1α and EPAS1/HIF-2α are hypoxia-inducible factors that share 48% sequence identity, they exhibit distinct expression patterns and functions in experimental models . HIF-1α has broader tissue distribution, whereas EPAS1 is predominantly expressed in highly vascularized tissues and endothelial cells . In experimental settings, EPAS1 shows relatively higher expression in placental villus sections and lung samples compared to other tissues . When designing experiments to study hypoxic responses, researchers should consider these differential expression patterns. Additionally, while both proteins bind to similar DNA elements, they may regulate different sets of target genes, necessitating specific experimental approaches when studying each factor individually.

What are the key characteristics of HRP-conjugated EPAS1 antibodies?

HRP-conjugated EPAS1 antibodies combine the target specificity of anti-EPAS1 antibodies with the enzymatic activity of horseradish peroxidase, eliminating the need for secondary antibodies in detection systems. Commercial HRP-conjugated EPAS1 antibodies like the EPAS-1/HIF-2 alpha Antibody (A-5) HRP typically have a concentration of 200 μg/ml . These conjugated antibodies are particularly valuable for Western blot applications where they can directly catalyze the oxidation of substrates like TMB to produce colorimetric signals, enhancing detection sensitivity and reducing experimental complexity. The HRP conjugation does not significantly alter the antibody's ability to recognize EPAS1, maintaining its specificity for nuclear and cytoplasmic localization in hypoxic conditions.

How should I optimize Western blot protocols for EPAS1 detection using HRP-conjugated antibodies?

For optimal Western blot detection of EPAS1 using HRP-conjugated antibodies, consider the following methodology based on validated protocols:

• Sample preparation: Use appropriate lysis buffers and include protease inhibitors to prevent EPAS1 degradation.

• Electrophoresis conditions: Run samples on a 5-20% SDS-PAGE gel at 70V (stacking gel) followed by 90V (resolving gel) for 2-3 hours .

• Loading quantity: Load approximately 30 μg of protein per lane under reducing conditions .

• Protein transfer: Transfer proteins to a nitrocellulose membrane at 150 mA for 50-90 minutes .

• Blocking: Block the membrane with 5% milk for 2 hours at room temperature .

• Primary antibody incubation: Incubate with HRP-conjugated EPAS1 antibody at a dilution of 1:1000 in 5% milk overnight at 4°C with rotation.

• Washing: Wash thoroughly with TBS-0.1% Tween (3 times, 5 minutes each) .

• Detection: Develop using a sensitive substrate system such as SuperSignal West Femto Maximum Sensitivity Substrate .

The expected band size for EPAS1 is approximately 120 kDa, though actual migration may vary slightly depending on post-translational modifications in different cell types or conditions .

What controls should be included when using EPAS1 antibodies in immunological detection methods?

When designing experiments with EPAS1 antibodies, comprehensive controls are essential for reliable data interpretation:

Including these controls allows for proper validation of results and helps troubleshoot potential issues in experimental procedures. For immunofluorescence applications, DAPI counterstaining helps verify nuclear localization of EPAS1 in hypoxic conditions as seen in DFO-treated HepG2 cells .

What are the recommended approaches for detecting EPAS1 in different types of tissue samples?

Different tissue types require tailored methodologies for optimal EPAS1 detection:

For paraffin-embedded tissue sections (such as prostate cancer or liver samples):

• Perform heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic .

• Apply primary EPAS1 antibody at concentrations of 0.3-3 μg/mL .

• For chromogenic detection, use appropriate secondary antibody systems such as Anti-Goat HRP-DAB for polyclonal antibodies or Anti-Rabbit IgG VisUCyte HRP Polymer for monoclonal antibodies .

• Counterstain with hematoxylin to visualize tissue architecture .

Specific staining should be localized to cytoplasm and nuclei in cancer cells or primarily to cell nuclei in liver tissue , depending on the physiological state and tissue type. Cellular localization data provides important information about EPAS1 activity, as nuclear translocation often indicates functional activation of this transcription factor.

How can I quantitatively measure EPAS1 protein levels in experimental samples?

For precise quantification of EPAS1 protein levels, researchers can employ sandwich ELISA methodology:

• Use a double antibody sandwich ELISA system with plates pre-coated with anti-EPAS1 capture antibody.

• Prepare a standard curve using recombinant EPAS1 protein in the range of 0.156-10 ng/ml .

• Add properly diluted samples (serum, plasma, cell culture supernatant, or tissue lysate) to the appropriate wells.

• Incubate with biotinylated detection antibody, which binds to EPAS1 captured by the immobilized antibody.

• Add HRP-Streptavidin Conjugate (SABC) and subsequently TMB substrate solution.

• Measure absorbance at 450 nm in a microplate reader after stopping the reaction.

• Calculate EPAS1 concentration using the standard curve .

This method offers high sensitivity (down to 0.094 ng/ml) and specificity for EPAS1 with minimal cross-reactivity. The entire procedure requires approximately 4 hours . For relative quantification in different experimental conditions, Western blot densitometry can be used as a complementary approach, particularly when comparing hypoxic versus normoxic conditions.

How do I troubleshoot weak or absent EPAS1 signal in Western blot applications?

When confronting weak or absent EPAS1 signals in Western blots, consider this systematic troubleshooting approach:

• Verify protein stabilization: EPAS1 is rapidly degraded in normoxic conditions. Treat cells with hypoxia-mimetic agents like DFO (1 mM, overnight) to stabilize EPAS1 protein .

• Optimize protein extraction:

  • Use nuclear extraction protocols, as EPAS1 predominantly localizes to the nucleus under hypoxic conditions.

  • Include proteasome inhibitors (MG132) to prevent degradation.

  • Maintain samples at 4°C throughout processing.

• Adjust antibody conditions:

  • Try different concentrations (1:500 to 1:2000 dilution range).

  • Extend primary antibody incubation time (overnight at 4°C).

  • Test alternative blocking agents if milk proteins interfere with detection.

• Enhance detection sensitivity:

  • Use femto-sensitivity chemiluminescent substrates for HRP detection.

  • Increase exposure time during imaging.

  • Consider signal amplification systems for low-abundance proteins.

• Verify antibody functionality with positive controls:

  • Include lysates from cells with known EPAS1 expression (e.g., DFO-treated HeLa cells) .

  • Confirm expected molecular weight (approximately 110-120 kDa) .

Remember that EPAS1 expression is highly regulated and may be undetectable under normal oxygen conditions without appropriate stabilization treatments.

What strategies can be employed to study EPAS1 interactions with other proteins in hypoxia response pathways?

To investigate EPAS1 protein interactions in hypoxia response pathways, researchers can implement several sophisticated methodologies:

• Co-immunoprecipitation (Co-IP):

  • Use EPAS1 antibodies for immunoprecipitation (like EPAS-1 Antibody (A-5) AC, agarose-conjugated) .

  • Analyze precipitated complexes for binding partners such as HIF-1β, VHL, or p300/CBP.

  • Include appropriate controls (IgG, normoxic vs. hypoxic conditions).

• Proximity ligation assay (PLA):

  • Detect in situ protein interactions with subcellular resolution.

  • Especially valuable for studying temporal dynamics of EPAS1 interactions during hypoxia.

• Chromatin immunoprecipitation (ChIP):

  • Investigate EPAS1 binding to hypoxia-responsive elements in target gene promoters.

  • Combine with sequencing (ChIP-seq) to identify novel EPAS1-regulated genes.

• Bimolecular fluorescence complementation (BiFC):

  • Visualize direct protein interactions in living cells.

  • Useful for mapping interaction domains between EPAS1 and other proteins.

• FRET-based approaches:

  • Monitor real-time interactions in living cells.

  • Particularly suitable for studying dynamic assembly/disassembly of EPAS1 complexes during oxygen fluctuations.

These methods can be combined with genetic approaches (mutation, deletion constructs) to map specific interaction domains and understand the functional significance of protein partnerships in hypoxia signaling pathways.

How can immunofluorescence be optimized to study EPAS1 nuclear translocation during hypoxia?

Immunofluorescence protocols can be optimized to visualize and quantify EPAS1 nuclear translocation during hypoxia:

• Cell preparation:

  • Culture cells on coverslips and expose to hypoxic conditions or hypoxia-mimetic agents (e.g., DFO).

  • Include both treated and untreated controls for comparison .

• Fixation and permeabilization:

  • Use immersion fixation to preserve cellular architecture.

  • Permeabilize cell membranes to allow antibody access to nuclear compartments.

• Antibody application:

  • Apply EPAS1 primary antibody at 3 μg/mL for 3 hours at room temperature .

  • Use fluorophore-conjugated secondary antibodies (e.g., NorthernLights 557-conjugated Anti-Rabbit IgG) .

• Nuclear counterstaining:

  • Counterstain with DAPI to visualize nuclei and facilitate assessment of EPAS1 nuclear localization .

• Quantitative analysis:

  • Capture images using confocal microscopy.

  • Quantify nuclear/cytoplasmic fluorescence intensity ratios.

  • Use automated image analysis software to assess multiple cells objectively.

In properly designed experiments, specific staining should be localized to nuclei in hypoxia-treated cells, while minimal or diffuse staining patterns appear in normoxic control cells . This differential localization reflects the oxygen-dependent regulation of EPAS1 stability and activity.

What approaches can be used to simultaneously detect multiple hypoxia-related factors alongside EPAS1?

To comprehensively analyze hypoxia response pathways, researchers often need to detect multiple factors simultaneously:

• Multiplex immunofluorescence:

  • Use primary antibodies from different host species (e.g., rabbit anti-EPAS1 and mouse anti-HIF-1α).

  • Apply spectrally distinct fluorophore-conjugated secondary antibodies.

  • Include additional markers such as GLUT1, VEGF, or PHD2 to assess downstream effects.

• Sequential immunohistochemistry:

  • For tissue sections, employ sequential detection with multiple chromogens.

  • For example, detect EPAS1 with DAB (brown) and other factors with alternative substrates.

  • This approach is particularly valuable for archival tissue samples.

• Integrated protein/mRNA detection:

  • Combine immunofluorescence for EPAS1 protein with RNA-FISH for target genes.

  • This correlation helps establish functional relationships between EPAS1 localization and transcriptional activity.

• Multiparametric flow cytometry:

  • For cell suspensions, use fluorophore-conjugated antibodies against EPAS1 and other factors.

  • Allows quantitative assessment of protein correlations at the single-cell level.

• Mass cytometry (CyTOF):

  • Enables simultaneous detection of numerous proteins using metal-tagged antibodies.

  • Particularly valuable for comprehensive profiling of hypoxia response networks.

These multiplexed approaches provide richer datasets that help unravel the complex interplay between different components of hypoxia signaling networks, offering insights that cannot be obtained from single-factor analyses.

How can EPAS1 detection be integrated with functional assays to study its role in cancer progression?

Integrating EPAS1 detection with functional assays provides mechanistic insights into its role in cancer progression:

• Correlative immunohistochemistry:

  • Detect EPAS1 in tumor tissue sections using protocols optimized for paraffin-embedded samples .

  • Correlate staining patterns with clinical parameters (stage, grade, patient outcomes).

  • Compare expression in matched normal and tumor tissues.

• Transcriptional reporter assays:

  • Use hypoxia-responsive element (HRE) luciferase reporters to assess EPAS1 transcriptional activity .

  • Compare reporter activation in cells with various levels of EPAS1 expression.

• Cell migration and invasion assays:

  • Manipulate EPAS1 expression (overexpression, knockdown) in cancer cell lines.

  • Measure changes in migratory and invasive capacity using Boyden chamber or wound healing assays.

  • Correlate findings with EPAS1 protein levels detected by Western blot or immunofluorescence.

• Angiogenesis assessment:

  • Evaluate EPAS1-dependent regulation of angiogenic factors (VEGF, angiopoietins).

  • Use tube formation assays with endothelial cells exposed to conditioned media from EPAS1-manipulated cancer cells.

  • Quantify microvessel density in xenograft models with varying EPAS1 expression.

• Metabolic profiling:

  • Assess glycolytic parameters in relation to EPAS1 expression.

  • Measure oxygen consumption rates and extracellular acidification.

  • Correlate metabolic profiles with EPAS1 protein levels determined by quantitative methods.

By combining these approaches, researchers can establish mechanistic links between EPAS1 expression, its transcriptional targets, and functional phenotypes relevant to cancer progression, particularly in contexts where hypoxia is a prominent feature of the tumor microenvironment.

How can phospho-specific antibodies be used to study post-translational regulation of EPAS1?

Post-translational modifications significantly influence EPAS1 function, and phospho-specific antibodies provide powerful tools to study these regulatory mechanisms:

• Key phosphorylation sites to target:

  • Thr-324 and Thr-487 (regulated by PKA, affecting transcriptional activity)

  • Ser-672 (regulated by CK1δ, influencing protein stability)

  • Tyr-674 (regulated by PDGFR, affecting nuclear localization)

• Experimental applications:

  • Western blot analysis comparing total EPAS1 versus phosphorylated forms

  • Immunoprecipitation followed by phospho-antibody detection

  • Immunofluorescence to track subcellular localization of phosphorylated EPAS1

• Kinase inhibitor studies:

  • Treat cells with specific kinase inhibitors

  • Monitor changes in EPAS1 phosphorylation status

  • Correlate with alterations in EPAS1 stability, localization, and transcriptional activity

• Mutational analysis:

  • Generate phospho-mimetic (S/T→D) or phospho-deficient (S/T→A) EPAS1 mutants

  • Validate phospho-antibody specificity using these mutants

  • Assess functional consequences of modification at specific sites

This approach provides mechanistic insights into how various signaling pathways converge on EPAS1 to fine-tune hypoxic responses in different cellular contexts and disease states.

What are the emerging approaches for studying EPAS1 in patient-derived samples and their clinical implications?

Emerging methodologies for EPAS1 analysis in patient samples offer promising avenues for translational research:

• Digital spatial profiling:

  • Analyze EPAS1 expression with spatial resolution in tumor microenvironments

  • Correlate with hypoxic gradients and other cell types in the tumor ecosystem

  • Identify spatial relationships between EPAS1-expressing cells and treatment-resistant regions

• Single-cell analysis:

  • Apply scRNA-seq and scATAC-seq to dissect EPAS1-dependent transcriptional programs

  • Identify rare cell populations with unique EPAS1 activation patterns

  • Characterize cellular heterogeneity in hypoxic response within tumors

• Liquid biopsy approaches:

  • Detect EPAS1 in circulating tumor cells or exosomes

  • Use sandwich ELISA methods with sensitivity down to 0.094 ng/ml

  • Develop as potential biomarkers for hypoxic tumors or treatment response

• Patient-derived organoids:

  • Establish 3D culture systems that maintain hypoxic gradients

  • Assess EPAS1 expression and function in these physiologically relevant models

  • Test targeted therapies against EPAS1-dependent pathways

• Predictive biomarker development:

  • Correlate EPAS1 expression patterns with treatment outcomes

  • Develop standardized scoring systems for EPAS1 immunohistochemistry

  • Create composite biomarker panels combining EPAS1 with other hypoxia markers

These approaches facilitate personalized medicine strategies by identifying patients likely to benefit from therapies targeting hypoxia-driven pathways in cancer and other diseases where EPAS1 plays a pivotal role.