HIF3A Antibody, Biotin conjugated

What is HIF3A and what cellular processes does it regulate?

HIF3A (Hypoxia-inducible factor 3-alpha) functions as a transcriptional regulator in adaptive responses to low oxygen tension. It acts as an inhibitor of angiogenesis in hypoxic cells of the cornea and plays a significant role in the development of the cardiorespiratory system . HIF3A is upregulated by hypoxia and exists in multiple alternatively spliced transcript variants . As part of the HIF family of transcription factors, HIF3A contributes to the cellular adaptation to hypoxic conditions, with particular importance in endothelial cell responses where HIF3A2 serves as a cell fate determinant during prolonged hypoxia .

What are the common applications for HIF3A antibody, biotin conjugated?

The biotin conjugation enhances detection sensitivity when used with streptavidin-based detection systems, making it particularly valuable for low-abundance protein detection scenarios .

How should HIF3A antibody, biotin conjugated be stored to maintain activity?

For optimal preservation of antibody activity, HIF3A antibody, biotin conjugated should be stored at -20°C or -80°C upon receipt . The antibody should be aliquoted to avoid repeated freeze-thaw cycles which can degrade protein structure and compromise binding efficacy. The typical storage buffer consists of preservative (0.03% Proclin 300) and constituents (50% Glycerol, 0.01M PBS, pH 7.4) , which helps maintain antibody stability during storage. The antibody remains stable for one year after shipment when properly stored .

What are the optimal cell models for studying HIF3A using biotin-conjugated antibodies?

Based on research findings, the following cell models are recommended when studying HIF3A with biotin-conjugated antibodies:

• HUVECs (Human Umbilical Vein Endothelial Cells): These cells predominantly express the HIF3A2 and HIF3A3 isoforms, with HIF3A2 being the major form expressed . They provide an excellent model for studying hypoxia responses in the vascular system.

• HeLa cells treated with Cobalt Chloride or Tunicamycin: These treatments induce hypoxic responses and increase HIF3A expression, making them suitable positive controls for antibody validation .

• A431 cells: These cells have been validated for positive HIF3A detection in Western blot applications .

When designing experiments, consider that HIF3A expression is induced under hypoxic conditions, so experimental protocols should include appropriate oxygen tension controls or hypoxia-mimicking agents like cobalt chloride for reliable results.

How can I validate the specificity of HIF3A antibody, biotin conjugated in my experimental system?

To validate antibody specificity, implement the following methodological approach:

• Positive and negative controls: Use cells known to express HIF3A (e.g., Cobalt Chloride-treated HeLa cells) as positive controls and include knockdown/knockout samples as negative controls. Published literature has utilized siRNA targeting HIF3A (Ambion assay id s34653) which targets three HIF3A isoforms - HIF3A variant 2 (NM_0224624), HIF3A variant 1 (NM_15794), and HIF3A variant 3 (NM_15279) .

• Antibody titration: Perform a titration experiment using the recommended dilution range (varies by application) to determine optimal antibody concentration for your specific experimental system.

• Competitive binding assay: Pre-incubate the antibody with recombinant HIF3A protein (such as the immunogen fragment, amino acids 516-669) to confirm binding specificity.

• Molecular weight verification: Confirm that the detected protein corresponds to the expected molecular weight of HIF3A (calculated: 72 kDa, observed: 72 kDa) .

• Cross-reactivity assessment: Test the antibody in multiple species if working with non-human models, noting that while the antibody is designed for human reactivity, some cross-reactivity with mouse samples has been cited .

How can I differentiate between HIF3A isoforms in experimental analysis?

Differentiating between HIF3A isoforms requires specialized methodological approaches:

• Isoform-specific primer design for RT-qPCR: Design primers that target unique regions of each HIF3A splice variant. For example, research has shown that in HUVECs, both HIF3A2 and HIF3A3 are expressed, with HIF3A2 being the predominant isoform .

• Western blotting with resolution optimization: Use lower percentage acrylamide gels (6-8%) to better separate higher molecular weight isoforms. Note that the commonly used rabbit anti-HIF-3α antibody (such as Sigma-Aldrich, AV39936) recognizes only the α1, α2, and α3 isoforms of HIF-3α .

• Isoform-specific siRNA knockdown: Employ targeted siRNA approaches to selectively knockdown specific isoforms and confirm antibody detection patterns. For example, in previous studies, HUVECs were transfected using Lipofectamine RNAiMax with siRNAs at a final concentration of 40 nM, and cells were cultured for 2 days prior to analysis .

• Recombinant expression of individual isoforms: Express individual HIF3A isoforms in a heterologous system as positive controls for antibody specificity testing.

What are the considerations for using HIF3A antibody, biotin conjugated in multi-color flow cytometry or immunofluorescence applications?

When implementing multi-color flow cytometry or immunofluorescence experiments with biotin-conjugated HIF3A antibodies, consider the following methodological approach:

• Fluorophore selection: Choose streptavidin conjugates with fluorophores that have minimal spectral overlap with other fluorophores in your panel.

• Signal amplification: Leverage the biotin-streptavidin interaction for signal amplification in cases where HIF3A expression is low or detection sensitivity is critical.

• Fixation and permeabilization optimization: Since HIF3A is a transcription factor with nuclear localization under hypoxic conditions, optimize your fixation and permeabilization protocol to ensure adequate nuclear penetration of the antibody. Published protocols have used tunicamycin-treated HeLa cells as positive controls for immunofluorescence applications .

• Compensation controls: Include single-stained controls for each fluorophore to enable proper compensation when using multiple fluorescent markers.

• Blocking of endogenous biotin: Pre-block endogenous biotin (particularly abundant in liver, kidney, and brain tissues) using streptavidin/avidin blocking kits to reduce background signal.

How can HIF3A antibody be used to investigate oxygen-dependent degradation mechanisms?

To study oxygen-dependent degradation of HIF3A, researchers can employ the following methodological strategies:

• ODD domain reporter assays: The HIF3A gene region comprising the oxygen-dependent degradation domain (amino acids 450-576) can be fused with firefly luciferase to create a reporter system. This approach has been validated in previous studies where the ODD domain was fused in-frame with firefly luciferase downstream of HIF3A, with renilla luciferase expressed from a different promoter as a transfection control .

• Proteasome inhibitor studies: Treat cells with proteasome inhibitors (e.g., MG132) under normoxic and hypoxic conditions to assess the contribution of proteasomal degradation to HIF3A regulation.

• Co-immunoprecipitation with VHL and PHD proteins: Use biotin-conjugated HIF3A antibodies in co-immunoprecipitation experiments to assess interactions with von Hippel-Lindau (VHL) protein and prolyl hydroxylase domain-containing proteins (PHDs) under varying oxygen conditions.

• Hydroxylation site mutation analysis: Generate hydroxylation site mutants of HIF3A and compare their stability to wild-type protein under normoxic and hypoxic conditions using the biotin-conjugated antibody for detection.

What are common issues when using HIF3A antibody, biotin conjugated and how can they be resolved?

How can I optimize HIF3A antibody, biotin conjugated for low abundance protein detection?

For detecting low abundance HIF3A protein, implement these optimization strategies:

• Signal amplification: Utilize multi-step detection with streptavidin-horseradish peroxidase (HRP) conjugates and enhanced chemiluminescence (ECL) substrates. Previous research has successfully employed SuperSignal West Pico ECL (Thermo Fisher Scientific) for HIF3A detection .

• Sample enrichment: Implement nuclear fractionation techniques to concentrate HIF3A protein, which predominantly localizes to the nucleus under hypoxic conditions.

• Hypoxia induction: Treat cells with hypoxia-mimicking agents such as cobalt chloride or culture in low oxygen conditions to upregulate HIF3A expression prior to analysis .

• Loading control optimization: When performing Western blots, use β-Actin (1:1000, ab1801; Abcam) as a reliable loading control as validated in published protocols .

• Blocking optimization: Use BSA (3% BSA, 0.5% Tween-20 for 1-2 hours) for blocking non-specific binding sites before antibody incubation .

How does HIF3A function differ from other HIF family members in hypoxic response pathways?

HIF3A has distinct functional characteristics compared to other HIF family members (HIF1A and HIF2A):

• Transcriptional regulation: While HIF1A and HIF2A primarily act as transcriptional activators, HIF3A often functions as an inhibitor of angiogenesis in hypoxic cells, particularly in the cornea . This inhibitory role suggests HIF3A may serve as a negative feedback regulator within the hypoxic response pathway.

• Isoform diversity: HIF3A has multiple alternatively spliced transcript variants with potentially different functions . For instance, in HUVECs, HIF3A2 is the predominant isoform expressed and serves as an endothelial cell fate determinant during prolonged hypoxia .

• Temporal expression patterns: HIF3A shows distinct temporal regulation compared to other HIF family members, with evidence suggesting its accumulation during prolonged hypoxia rather than acute hypoxic responses, indicating a role in adaptive rather than immediate hypoxic responses .

• Tissue specificity: HIF3A demonstrates tissue-specific expression patterns and roles, with significant involvement in the development of the cardiorespiratory system .

Research using HIF3A antibodies, including biotin-conjugated formats, can help elucidate these functional differences through comparative expression studies, ChIP-seq analyses of binding targets, and knockdown/overexpression experiments examining differential gene regulation.

What role does HIF3A play in endothelial cell responses to chronic hypoxia?

Research using HIF3A antibodies has revealed that HIF3A2 accumulation is a critical component of human endothelial cell response to prolonged hypoxia . Methodological investigations have shown:

• Temporal regulation: Unlike the rapid and transient induction of HIF1A, HIF3A2 accumulation occurs during extended hypoxic conditions, suggesting a role in long-term adaptation rather than acute response .

• Cell fate determination: HIF3A2 serves as an endothelial cell fate determinant during prolonged hypoxia, influencing cellular processes beyond immediate metabolic adaptations .

• Expression mechanism: The accumulation of HIF3A2 results from both transcriptional and post-translational mechanisms. This has been demonstrated using reporter assays where the HIF3A gene region comprising the oxygen-dependent degradation domain (amino acids 450-576) was fused with firefly luciferase .

• Isoform specificity: In HUVECs, only HIF3A2 and HIF3A3 are expressed, with HIF3A2 being the predominant form. This isoform specificity suggests unique roles for different HIF3A variants in endothelial responses to hypoxia .

To further investigate these mechanisms, researchers can employ biotin-conjugated HIF3A antibodies in ChIP-seq experiments to identify genomic binding sites and regulatory targets specific to endothelial cells under chronic hypoxic conditions.