EPAS1 Antibody, Biotin conjugated

What is EPAS1 and what are its primary biological functions?

EPAS1, also known as HIF2α (Hypoxia-Inducible Factor 2-alpha), is an 870 amino acid protein transcription factor involved in oxygen-regulated gene expression. It is primarily expressed in most tissues, with highest expression levels in placenta, lung, and heart. EPAS1 functions by binding to core DNA sequence 5'-[AG]CGTG-3' within the hypoxia response element (HRE) of target gene promoters . As a key regulator of cellular response to hypoxia, EPAS1 controls vascular endothelial growth factor (VEGF) expression and appears critical for blood vessel development and the tubular system of the lung . Recent research has also demonstrated EPAS1's crucial role in spermatogonial stem cell function, particularly under regenerative conditions .

What is the molecular structure of EPAS1 and how does this impact antibody binding?

EPAS1 has a calculated molecular weight of 96 kDa, though it is typically observed at 100-120 kDa on Western blots due to post-translational modifications . The protein colocalizes with HIF3A in the nucleus and nuclear speckles. When developing research strategies using EPAS1 antibodies, it is important to consider that EPAS1 stability is oxygen-dependent - the protein is degraded at oxygen concentrations >5% O₂ . This oxygen sensitivity must be accounted for in experimental design, particularly when working with samples that may experience varying oxygen levels during processing.

What are the optimal experimental conditions for using biotin-conjugated EPAS1 antibodies?

For optimal results with biotin-conjugated EPAS1 antibodies, researchers should consider the following conditions:

When working with biotin-conjugated antibodies, researchers should titrate the antibody concentration for their specific experimental system to obtain optimal signal-to-noise ratios . Additionally, consideration of endogenous biotin in certain tissues (particularly liver, kidney, and brain) is essential, with appropriate blocking steps implemented to minimize background.

How should hypoxic conditions be replicated when studying EPAS1 expression?

Since EPAS1 is a hypoxia-inducible factor, proper experimental design for studying its expression patterns requires careful control of oxygen conditions. Researchers can use cobalt chloride treatment (as demonstrated in HeLa cells) as a chemical inducer of hypoxia, or utilize controlled oxygen chambers to maintain physiologically relevant hypoxic conditions (typically 1-5% O₂). For instance, in studies of spermatogonial stem cells, researchers maintained cultures at 10% O₂ in 5% CO₂ incubators to study EPAS1 expression .

Alternative approaches include using prolyl-hydroxylase inhibitors such as Daprodustat, which has been shown to induce a dose-dependent increase in EPAS1 expression without affecting HIF1A levels or cell viability . This pharmacological approach offers precise temporal control over EPAS1 stabilization without requiring specialized hypoxia chambers.

What controls are essential when validating EPAS1 antibody specificity?

When validating biotin-conjugated EPAS1 antibodies, several controls are critical:

• Positive Controls: Utilize cobalt chloride-treated HeLa cells, which demonstrate robust EPAS1 expression .

• Negative Controls: Include samples from EPAS1 knockout models or cells treated with EPAS1-specific siRNA.

• Blocking Peptide Controls: Pre-incubate the antibody with the immunogen peptide to confirm signal specificity.

• Isotype Controls: Include an irrelevant biotin-conjugated antibody of the same isotype (Rabbit IgG) to assess non-specific binding.

• Endogenous Biotin Controls: Include streptavidin-detection reagent alone (without primary antibody) to evaluate endogenous biotin background, particularly in biotin-rich tissues.

When performing multiplexed experiments, additional controls to assess potential cross-reactivity with other antibodies in the panel are essential for accurate data interpretation.

How can researchers address variable EPAS1 detection across different experimental conditions?

Inconsistent EPAS1 detection often stems from the protein's oxygen sensitivity and various regulatory mechanisms. To address this variability:

• Standardize Sample Handling: Process all samples under identical conditions, ideally in a hypoxic environment or with rapid processing to prevent oxygen-dependent degradation.

• Control Oxygen Exposure: Maintain consistent oxygen levels during sample collection and processing, as EPAS1 is degraded at >5% O₂ .

• Consider Post-Translational Modifications: EPAS1 undergoes hydroxylation, ubiquitination, and other modifications that affect antibody recognition. The observed molecular weight (100-120 kDa) differs from the calculated weight (96 kDa) due to these modifications .

• Evaluate Expression Patterns: EPAS1 expression varies significantly across cell types, even within the same tissue. For example, studies show that 95% of spermatogonial stem cells (SSCs) are EPAS1+, compared to only 6.5% of progenitor cells . This heterogeneity must be considered when interpreting results.

• Account for Genetic Variations: Polymorphisms in the EPAS1 promoter can significantly alter expression levels. The rs56721780:G>C variant and the presence/absence of a 40-bp insertion fragment at position -742 impact transcriptional regulation .

What strategies can minimize background when using biotin-conjugated antibodies for EPAS1 detection?

Background signal is a common challenge with biotin-conjugated antibodies. To minimize this issue:

• Block Endogenous Biotin: Use commercial biotin blocking kits or a sequential blocking approach with free avidin followed by free biotin before applying the biotin-conjugated primary antibody.

• Optimize Antibody Concentration: Titrate the biotin-conjugated EPAS1 antibody to determine the minimal concentration needed for specific signal detection.

• Use Appropriate Buffers: Include proteins like BSA (0.1-1%) and detergents like Tween-20 (0.05-0.1%) in washing and blocking buffers to reduce non-specific binding.

• Consider Tissue Autofluorescence: When using fluorescent streptavidin conjugates for detection, implement autofluorescence reduction strategies such as Sudan Black B treatment or spectral unmixing during image acquisition.

• Evaluate Fixation Impact: Different fixation methods can affect endogenous biotin levels and accessibility. Compare paraformaldehyde, methanol, and acetone fixation to determine optimal conditions for your specific sample type.

How can EPAS1 antibodies be utilized to investigate hypoxia signaling networks?

Advanced investigation of hypoxia signaling networks using EPAS1 antibodies involves several sophisticated approaches:

• Chromatin Immunoprecipitation (ChIP): Biotin-conjugated EPAS1 antibodies can be used in ChIP assays to identify direct genomic binding sites. This approach revealed that EPAS1 binds to the core DNA sequence 5'-[AG]CGTG-3' within hypoxia response elements .

• Co-Immunoprecipitation Studies: To elucidate protein interaction networks, researchers can use EPAS1 antibodies to identify binding partners under various oxygen conditions. This has helped establish relationships between EPAS1 and other hypoxia response factors.

• Proximity Ligation Assays (PLA): This technique enables visualization of protein-protein interactions in situ, allowing researchers to map EPAS1 interactions within cellular microenvironments.

• Transcriptional Reporter Systems: Combined with EPAS1 antibody validation, reporter constructs containing hypoxia response elements can quantify transcriptional activity under various conditions, correlating with EPAS1 protein levels detected by antibody-based methods.

• Single-Cell Analysis: Advanced imaging using biotin-conjugated EPAS1 antibodies with multiplexed detection systems can reveal cell-to-cell variability in hypoxic responses within heterogeneous tissues.

How can researchers study the relationship between EPAS1 genetic variants and protein expression?

Research on EPAS1 genetic variants and their impact on protein expression requires integrated methodological approaches:

• Promoter Activity Analysis: The EPAS1 promoter contains functional polymorphisms that affect expression. For example, the rs56721780:G>C variant affects binding of the transcription factor IKZF1, with the G allele showing stronger binding than the C allele . Luciferase reporter assays combined with antibody-based protein quantification can correlate genotype with expression levels.

• Allele-Specific Expression: Using biotin-conjugated antibodies in conjunction with genotyping can reveal whether certain EPAS1 variants show differential protein expression patterns.

• Haplotype Analysis: Studies have shown that specific haplotypes (e.g., G/deletion/C versus C/insertion/T) correlate with different EPAS1 mRNA and protein levels. The C/insertion/T haplotype is associated with higher expression levels, potentially due to enhanced Sp1 binding to the 40-bp insertion fragment .

• Functional EMSA Studies: Electrophoretic mobility shift assays have confirmed differential binding of transcription factors to variant EPAS1 promoter sequences. For example, Sp1 binds to the 40-bp insertion fragment, acting as a transcriptional activator .

• Population Genetics: EPAS1 variants have been linked to high-altitude adaptation, making antibody-based studies of expression patterns across different genetic backgrounds particularly valuable for understanding evolutionary adaptations to hypoxia.

What methodological considerations are important when using EPAS1 antibodies to study stem cell biology?

When applying EPAS1 antibodies to stem cell research, several specialized methodological considerations emerge:

• Oxygen Sensitivity of Stem Cell Niches: Studies have shown that spermatogonial stem cells (SSCs) reside in hypoxic microenvironments, with 95% of SSCs expressing EPAS1 . Researchers must maintain appropriate oxygen conditions during sample processing to preserve physiological EPAS1 expression.

• Stem Cell Heterogeneity: Within stem cell populations, EPAS1 expression can vary significantly. For example, 95% of ID4-EGFP-labeled SSCs were EPAS1+, compared to only 6.5% of progenitors . Antibody-based flow cytometry or imaging must account for this heterogeneity.

• Culture Adaptation Effects: Prolonged culture of stem cells can alter EPAS1 expression. Studies showed significant decline in EPAS1 expression when comparing spermatogonia maintained in culture (at 5% CO₂, 10% O₂) versus those isolated directly from the testis .

• Pharmacological Modulation: Prolyl-hydroxylase inhibitors like Daprodustat can be used to stabilize EPAS1 in cultured stem cells, producing a dose-dependent increase in EPAS1 expression without affecting cell viability . This approach allows controlled experimental manipulation of EPAS1 levels.

• Regenerative Capacity Assessment: Transplantation experiments have shown that EPAS1 expression impacts stem cell regenerative function. EPAS1 was found to be required for SSC function in regenerative conditions post-chemotherapy treatment . Antibody-based tracking of EPAS1 expression can help predict regenerative potential.

How can cutting-edge imaging techniques enhance EPAS1 detection with biotin-conjugated antibodies?

Advanced imaging technologies offer new opportunities for EPAS1 detection and functional analysis:

• Super-Resolution Microscopy: Techniques like STORM or PALM, when combined with biotin-conjugated EPAS1 antibodies and fluorescent streptavidin, enable visualization of EPAS1 distribution at nanometer resolution, revealing previously undetectable spatial organization within the nucleus.

• Live-Cell Imaging: Novel membrane-permeable biotin-conjugated antibody fragments allow tracking of EPAS1 dynamics in living cells under changing oxygen conditions, providing temporal information about protein stabilization and degradation.

• Tissue Clearing Methods: Whole-organ or tissue-clearing techniques (CLARITY, iDISCO) combined with biotin-conjugated EPAS1 antibodies enable three-dimensional visualization of EPAS1 expression patterns throughout entire organs, revealing spatial relationships within complex tissues.

• Correlative Light-Electron Microscopy: This approach allows researchers to correlate fluorescent signals from biotin-streptavidin detection with ultrastructural features, providing context for EPAS1 localization at the subcellular level.

• Multiplexed Imaging: Cyclic immunofluorescence or mass cytometry techniques enable simultaneous detection of EPAS1 alongside dozens of other proteins, creating comprehensive maps of hypoxia response pathways in complex tissues.

What are the most promising RNA-protein correlation approaches for validating EPAS1 antibody specificity?

Integrated RNA-protein analysis provides powerful validation for antibody specificity:

• Single-Cell Multi-Omics: Combined single-cell RNA sequencing and protein detection (CITE-seq or REAP-seq) with biotin-conjugated EPAS1 antibodies allows direct correlation between EPAS1 transcript and protein levels at single-cell resolution.

• Spatial Transcriptomics: Technologies like Visium or MERFISH combined with immunofluorescence using biotin-conjugated EPAS1 antibodies enable spatial correlation of EPAS1 mRNA and protein distribution in tissue sections.

• Translating Ribosome Affinity Purification (TRAP): This technique can isolate actively translating EPAS1 mRNA, which can be correlated with protein levels detected by antibodies to understand translational regulation under hypoxic conditions.

• RNA-Protein Correlation in Genetic Studies: RNA-seq studies demonstrated that specific EPAS1 haplotypes (e.g., G/deletion/C versus C/insertion/T) correlate with different mRNA levels, with individuals carrying two copies of the C/insertion/T haplotype showing much higher EPAS1 mRNA levels . These findings can validate antibody-detected protein variations.

• Inducible Expression Systems: Controlled expression of EPAS1 through inducible systems allows precise correlation between induced mRNA and the resulting protein detection by antibodies, serving as a gold-standard validation approach.