HIF3A Antibody, FITC conjugated

What is HIF3A and what is its primary biological function?

HIF3A (Hypoxia-inducible factor 3-alpha) acts as a transcriptional regulator in adaptive response to low oxygen tension. It functions as an inhibitor of angiogenesis in hypoxic cells of the cornea and plays a crucial role in the development of the cardiorespiratory system. Unlike other HIF family members, HIF-3α and its variant NEPAS are less transcriptionally active than HIF-1α and HIF-2α because they lack a C-terminal transcriptional activation domain (C-TAD). This structural difference enables HIF-3α and NEPAS to repress the activity of HIF-1α or HIF-2α when dimerized with HIF-1β. The HIF3A protein is significantly up-regulated under hypoxic conditions, functioning as part of the cellular oxygen-sensing mechanism .

What applications is the HIF3A Antibody, FITC conjugated suitable for?

The FITC-conjugated HIF3A antibody has been validated for multiple research applications:

The antibody has been successfully used in various experimental systems, particularly showing strong signals in cobalt chloride-treated HeLa cells and A431 cells for Western blot applications, and in tunicamycin-treated HeLa cells for immunofluorescence studies .

What are the optimal storage conditions for HIF3A Antibody, FITC conjugated?

For maximum stability and retention of fluorescence activity, FITC-conjugated HIF3A antibody should be stored at -20°C or -80°C and protected from light exposure. The antibody is typically supplied in a storage buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, or similar preservation solutions (some variants use 0.03% Proclin 300). Under these conditions, the antibody remains stable for at least one year after shipment. Repeated freeze-thaw cycles should be avoided to maintain antibody integrity. For -20°C storage, aliquoting is generally unnecessary for the standard preparation .

What species reactivity does the HIF3A Antibody, FITC conjugated demonstrate?

The HIF3A Antibody has been tested and confirmed to react with human samples. Published studies have also cited reactivity with mouse samples, though human reactivity is more extensively documented. When selecting this antibody for your research, consider that it is typically generated using recombinant human Hypoxia-Inducible Factor 3-Alpha protein (commonly using the 516-669AA region as immunogen) and has been affinity purified to ensure specificity .

How do I optimize immunofluorescence protocols for HIF3A Antibody, FITC conjugated?

For optimal immunofluorescence results with FITC-conjugated HIF3A antibody:

• Sample preparation: Induce HIF3A expression with appropriate stimuli such as cobalt chloride or tunicamycin treatment in cell cultures, as these have been validated to increase HIF3A expression.

• Fixation optimization: Test both paraformaldehyde (4%) and methanol fixation methods, as HIF transcription factors can be sensitive to fixation conditions.

• Dilution series: Begin with the recommended dilution range (1:200-1:800) and perform a titration to determine optimal signal-to-noise ratio for your specific sample.

• Counterstaining: Use DAPI for nuclear visualization, as HIF3A can shuttle between nucleus and cytoplasm depending on oxygen conditions.

• Controls: Include both positive controls (tunicamycin-treated HeLa cells) and negative controls (secondary antibody only) to validate specificity.

• Photobleaching prevention: Minimize exposure to light during all steps of the protocol and mount samples with anti-fade mounting medium to preserve FITC fluorescence .

What are the most effective methods for validating HIF3A antibody specificity?

Rigorous validation of HIF3A antibody specificity is essential for reliable research outcomes:

• Genetic approach: Use HIF3A gene-disrupted cells/tissues as negative controls. Research has demonstrated that HIF3A gene-disrupted mice (generated through genome editing) provide excellent negative controls for antibody specificity testing .

• Knockdown validation: Perform siRNA or shRNA knockdown of HIF3A and confirm reduced signal in Western blot and immunofluorescence applications.

• Blocking peptide: Pre-incubate the antibody with the immunizing peptide to confirm that this eliminates specific binding.

• Cross-reactivity assessment: Test against related proteins (HIF1A, HIF2A) to ensure the antibody doesn't recognize these structurally related proteins.

• Molecular weight verification: Confirm that the observed band in Western blot matches the expected molecular weight of HIF3A (72 kDa) .

How can I effectively use HIF3A Antibody, FITC conjugated to study hypoxia-related pathways?

To leverage FITC-conjugated HIF3A antibody for hypoxia pathway research:

• Comparative analysis: Design experiments that compare HIF3A expression/localization with HIF1A and HIF2A under varying oxygen tensions to understand their differential regulation.

• Time-course studies: Monitor HIF3A expression at different time points after hypoxia induction to understand temporal dynamics.

• Co-immunoprecipitation: Use the antibody (non-FITC version) for co-IP experiments to identify novel interaction partners of HIF3A under hypoxic conditions.

• Tissue-specific expression: Investigate HIF3A expression in different tissues, particularly focusing on lung tissue where HIF3A has been implicated in development.

• Correlative microscopy: Combine immunofluorescence with other imaging modalities to correlate HIF3A expression with physiological parameters like local oxygen concentration or metabolic activity .

What considerations are important when designing co-localization experiments with HIF3A Antibody, FITC conjugated?

When designing co-localization studies:

• Spectral separation: Since the antibody is FITC-conjugated (green fluorescence), select compatible fluorophores for other targets that have minimal spectral overlap (such as Cy3, Cy5, or Alexa 594/647).

• Fixation consistency: Use consistent fixation methods for all antibodies in the multiplex staining protocol to ensure comparable antigen accessibility.

• Sequential staining: Consider sequential rather than simultaneous staining if using multiple rabbit-derived antibodies to prevent cross-reactivity.

• Relevant co-localization targets: Design experiments to examine co-localization with:

  • HIF1β (ARNT) - the obligate dimerization partner

  • Other hypoxia-responsive proteins

  • Nuclear markers to assess nuclear translocation under hypoxia

  • Proteasomal components to study degradation kinetics

• Positive controls: Include known co-localizing proteins as technical validation .

How can I resolve weak or no signal when using HIF3A Antibody, FITC conjugated?

When experiencing weak or absent signal:

• Antigen retrieval optimization: If performing IHC or IF on fixed tissues, test different antigen retrieval methods (heat-induced vs. enzymatic) to improve epitope accessibility.

• Hypoxia induction: HIF3A expression is highly oxygen-dependent. Ensure your experimental system includes proper hypoxic conditions (1-5% O₂) or chemical hypoxia mimetics like cobalt chloride to induce expression.

• Antibody concentration: Increase antibody concentration gradually, with careful titration from 1:200 to 1:100 if necessary, while monitoring background.

• Signal amplification: Consider using a biotin-streptavidin system or tyramide signal amplification if signal strength remains insufficient.

• Protein degradation prevention: Add proteasome inhibitors (MG132) to your sample preparation protocol, as HIF proteins are rapidly degraded under normoxic conditions.

• Expression verification: Confirm HIF3A expression in your specific sample type through RT-PCR before immunostaining attempts .

What controls should I include when working with HIF3A Antibody, FITC conjugated?

Robust experimental design requires appropriate controls:

• Positive tissue/cell control: Include cobalt chloride-treated HeLa cells or A431 cells, which have been confirmed to express detectable levels of HIF3A.

• Negative controls:

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

  • Isotype control (rabbit IgG-FITC) to evaluate background

  • HIF3A knockout or knockdown samples when available

• Competing peptide control: Pre-absorb the antibody with immunizing peptide to demonstrate binding specificity.

• Normoxia vs. hypoxia comparison: Include both conditions to demonstrate oxygen-dependent regulation.

• Non-conjugated primary antibody control: Compare results with non-FITC conjugated version to ensure conjugation doesn't affect specificity .

How do I accurately quantify fluorescence intensity in HIF3A immunostaining experiments?

For reliable quantification of HIF3A immunofluorescence:

• Image acquisition standardization: Use identical exposure settings, gain, and offset for all experimental groups.

• Background subtraction: Implement consistent background subtraction based on negative control samples.

• Region of interest selection: Define anatomical or cellular regions objectively based on nuclear counterstain or cellular markers rather than the HIF3A signal itself.

• Z-stack analysis: For thick specimens, collect z-stacks and perform maximum intensity projections or 3D analysis.

• Software tools: Use specialized image analysis software (ImageJ/FIJI with appropriate plugins, CellProfiler, etc.) for:

  • Nuclear vs. cytoplasmic signal ratio analysis

  • Mean fluorescence intensity measurements

  • Colocalization coefficients when performing dual labeling

• Normalization approaches: Normalize to cell number, tissue area, or housekeeping protein expression to account for variation in cell density .

What could cause unexpected cellular localization patterns of HIF3A?

Aberrant localization patterns may result from:

• Splice variant expression: HIF3A has multiple splice variants (including IPAS) with different subcellular localizations. The antibody may detect multiple variants depending on the epitope recognized.

• Oxygen-independent regulation: Unlike HIF1A, HIF3A localization can be regulated by factors beyond oxygen tension, including certain growth factors and inflammatory signals.

• Cell type-specific mechanisms: Different cell types may exhibit unique HIF3A regulation and localization patterns.

• Fixation artifacts: Overfixation can cause artificial translocation or mask true localization patterns.

• Post-translational modifications: Phosphorylation, SUMOylation, or other modifications may affect nuclear-cytoplasmic shuttling.

• Experimental conditions: Cell confluency, serum components, or culture medium composition can influence HIF3A expression and localization .

How can I use HIF3A Antibody, FITC conjugated to study the role of HIF3A in lung development?

For lung development studies:

• Developmental time course: Design immunofluorescence experiments examining HIF3A expression across different developmental stages, particularly during alveolarization phases.

• Comparative histology: Study control versus HIF3A-deficient tissues to correlate protein expression with structural changes. Research has shown that HIF3A gene-disrupted mice exhibit abnormal configuration of the lung, including reduced number of alveoli and thickened alveolar walls.

• Co-expression analysis: Investigate co-expression with surfactant proteins, alveolar markers, and vascular development factors using multiplex immunofluorescence.

• In vitro models: Implement air-liquid interface cultures of lung epithelial cells to study HIF3A regulation during differentiation.

• Hypoxia challenges: Examine how altered oxygen tensions during development affect HIF3A expression patterns and subsequent developmental outcomes.

• Genetic interaction studies: Combine with examination of other HIF family members to understand compensatory mechanisms, as HIF3A functions differ from HIF1A and HIF2A in developmental contexts .

What approaches can reveal interactions between HIF3A and other HIF family members?

To investigate HIF family member interactions:

• Co-immunoprecipitation: Use non-conjugated HIF3A antibody for pull-down experiments followed by detection of other HIF family members.

• Proximity ligation assay: Combine HIF3A antibody with antibodies against HIF1A, HIF2A, or HIF1β to visualize and quantify in situ protein interactions with subcellular resolution.

• Chromatin immunoprecipitation (ChIP): Examine competitive binding to hypoxia response elements by different HIF complexes.

• Sequential ChIP: Determine if HIF3A and other HIF family members co-occupy the same genomic regions.

• FRET or BRET analysis: For live-cell studies of protein interactions, though this requires fluorescent protein tagging rather than antibody labeling.

• Transcriptomic analysis: Compare gene expression changes in knockdowns of individual HIF family members versus combination knockdowns to identify synergistic effects .

How can HIF3A Antibody, FITC conjugated be optimized for flow cytometry applications?

To optimize flow cytometry protocols:

• Cell preparation: Use gentle fixation (2% paraformaldehyde) and permeabilization (0.1% Triton X-100 or saponin) to preserve cellular integrity while allowing antibody access.

• Compensation controls: Prepare single-color controls for each fluorophore in your panel to properly compensate for spectral overlap, particularly important when using FITC alongside other green-yellow fluorophores.

• Titration optimization: Perform a detailed titration of the FITC-conjugated HIF3A antibody to determine the concentration that provides maximum signal-to-noise ratio.

• Gating strategy: Develop a gating strategy that accounts for cell cycle phases, as HIF expression can vary with cell cycle position.

• Viability discrimination: Include a viability dye compatible with FITC to exclude dead cells that may show non-specific antibody binding.

• Hypoxic induction: For positive controls, include samples from cells cultured under hypoxic conditions or treated with hypoxia-mimetic agents like cobalt chloride to ensure detectable HIF3A expression .

What are the emerging implications of HIF3A in metabolic research?

HIF3A has emerging significance in metabolic regulation:

• Adipose tissue studies: Recent research indicates HIF3A involvement in the metabolic processes of fat tissue. The FITC-conjugated antibody can be used to examine HIF3A expression in adipocytes under different metabolic conditions.

• Epigenetic regulation: Investigate methylation patterns of the HIF3A gene in metabolic disorders using the antibody to correlate protein expression with epigenetic modifications.

• Nutrient sensing pathways: Examine interactions between HIF3A and nutrient sensing pathways such as mTOR using co-localization studies.

• Metabolic stress responses: Study HIF3A expression and localization changes during various metabolic challenges (glucose deprivation, lipid overload, etc.).

• Tissue-specific metabolic functions: Compare HIF3A expression patterns across metabolically active tissues like liver, muscle, and adipose to understand tissue-specific functions.

• Intervention studies: Use the antibody to track changes in HIF3A expression following metabolic interventions such as caloric restriction or exercise regimens .

What are the detailed specifications of commercially available HIF3A Antibody, FITC conjugated?

The FITC-conjugated HIF3A antibody has the following technical specifications:

These specifications ensure consistent performance across experimental applications and facilitate proper experimental design and troubleshooting .

How should I validate lot-to-lot consistency when using HIF3A Antibody, FITC conjugated?

To ensure experimental reproducibility across antibody lots:

• Positive control standardization: Maintain a standard positive control (e.g., lysate from cobalt chloride-treated cells) that can be used to compare antibody performance between lots.

• Quantitative metrics: Document key performance indicators for each lot:

  • Signal-to-noise ratio in immunofluorescence

  • Band intensity and specificity in Western blot

  • Titration curves in flow cytometry

• Reference sample archiving: Preserve reference samples (fixed cells or tissue sections) that worked well with previous lots for side-by-side comparison.

• Fluorescence intensity calibration: Use calibration beads to standardize FITC fluorescence intensity measurements across different experimental sessions.

• Documentation: Maintain detailed records of antibody performance, including images and quantitative data, to identify any lot-specific variations.

• Epitope verification: If possible, confirm that different lots recognize the same epitope region through peptide competition assays .