HIF1AN Antibody

What is HIF1AN and what is its function in cellular oxygen sensing?

HIF1AN (also known as FIH-1) functions as an oxygen sensor in cells by hydroxylating HIF-1 alpha at 'Asn-799' in the C-terminal transactivation domain (CAD). Under normoxic conditions, this hydroxylation prevents interaction of HIF-1 with transcriptional coactivators including Cbp/p300-interacting transactivator, effectively inhibiting hypoxia-responsive gene expression . Beyond its role in HIF regulation, HIF1AN is involved in transcriptional repression through interaction with HIF1A, VHL and histone deacetylases. It also hydroxylates specific Asn residues within ankyrin repeat domains (ARD) of multiple proteins including NFKB1, NFKBIA, NOTCH1, ASB4, and PPP1R12A . Additionally, it can hydroxylate Asp and His residues within ARDs of ANK1 and TNKS2, respectively, demonstrating its diverse role in post-translational modifications .

How can I detect HIF1AN in different cell and tissue types?

HIF1AN can be detected in various cell and tissue types using specific antibodies optimized for different applications. Western blot analysis has successfully detected HIF1AN in multiple human cell lines including HEK-293, HeLa, and Jurkat cells . For mouse samples, HIF1AN has been detected in brain, heart, skeletal muscle, hypothalamus, and colon tissue lysates . Rat samples showing HIF1AN expression include brain and heart tissue lysates .

Immunohistochemistry has successfully detected HIF1AN in human lung cancer tissue using antigen retrieval with TE buffer pH 9.0 or alternatively with citrate buffer pH 6.0 . For fluorescent immunocytochemistry, HIF1AN can be visualized in both cytoplasm and nuclei of human cell lines such as A172 glioblastoma cells, as well as in the cytoplasm of C2C12 mouse myoblast cells . When designing your experiment, consider that expression levels may vary between tissues, so optimization of antibody concentration and detection methods may be necessary for each specific sample type.

What are the common molecular weights observed for HIF1AN in Western blot applications?

HIF1AN is consistently observed at approximately 40-42 kDa in Western blot applications across different antibodies and sample types. Specifically:

• R&D Systems antibody (MAB7874) detects HIF1AN at approximately 42 kDa in A172 human glioblastoma and C2C12 mouse myoblast cell lines under reducing conditions .

• Abcam's antibody (ab307829) shows a band of 42 kDa in various mouse and rat tissue lysates .

• Bio-Rad's PrecisionAb antibody (clone HIF 162c) detects a band of approximately 42 kDa in HEK293 cell lysate .

• Proteintech's antibody (10646-1-AP) observes the protein at its calculated molecular weight of 40 kDa .

These consistent findings across different antibodies and sample types confirm the expected molecular weight range for HIF1AN detection. When troubleshooting your Western blot, expect to see the primary band between 40-42 kDa depending on your specific sample and detection system.

What are the optimal conditions for Western blot detection of HIF1AN?

For optimal Western blot detection of HIF1AN, consider the following methodological parameters based on validated protocols:

PVDF membranes are commonly used for HIF1AN Western blot applications . For reducing conditions, several immunoblot buffer systems have been validated, with Immunoblot Buffer Group 1 being specifically mentioned in some protocols . The recommended antibody dilutions vary by manufacturer but typically range from 1:500 to 1:2000 . R&D Systems antibody MAB7874 was effective at 0.25 μg/mL , while Abcam's antibody ab307829 performed well at 1:1000 dilution .

For protein loading, 20 μg of lysate is commonly used for tissue samples , while cell line lysates have been tested at concentrations ranging from 0.2-0.5 mg/mL on Simple Western systems . Secondary antibody detection typically employs HRP-conjugated anti-mouse or anti-rabbit IgG depending on the primary antibody host species, with dilutions ranging from 1:10,000 to 1:100,000 .

When troubleshooting, consider that some antibodies may show non-specific interactions with standards in automated Western systems like Simple Western, as noted with MAB7874 .

How can I validate the specificity of HIF1AN antibodies?

Validating antibody specificity is crucial for generating reliable data. Based on the search results, several approaches have been employed to confirm HIF1AN antibody specificity:

• Knockout cell line validation: Western blot comparison between parental and knockout cell lines provides the strongest evidence of specificity. For example, HIF1AN antibodies have been validated using HIF1AN knockout 293T cells compared with wild-type 293T cells . The absence of the expected 42 kDa band in knockout cells confirms antibody specificity.

• siRNA knockdown validation: C2C12 cells transfected with siRNA specifically targeting HIF1AN/FIH-1 showed reduced band intensity compared to control samples when probed with anti-HIF1AN antibody .

• Cross-species reactivity assessment: Testing the antibody against samples from multiple species (human, mouse, rat) can help establish conservation of the epitope and antibody utility across experimental models .

• Multiple antibody comparison: Using different antibodies targeting distinct epitopes of HIF1AN and observing consistent results strengthens confidence in specificity.

• Subcellular localization: Immunocytochemical localization of HIF1AN to expected compartments (cytoplasm and/or nuclei) provides additional validation .

When designing your validation strategy, consider incorporating at least two of these approaches to establish confident specificity of your selected HIF1AN antibody.

What controls should be included when working with HIF1AN antibodies?

Proper experimental controls are essential for interpreting HIF1AN antibody results. Based on the search results, the following controls should be considered:

• Positive controls: Use cell lines with known HIF1AN expression such as A172, HEK-293, HeLa, Jurkat, or C2C12 cells . Tissue lysates from brain, heart, skeletal muscle, hypothalamus, or colon can also serve as positive controls .

• Negative controls: HIF1AN knockout cell lines provide the ideal negative control. The search results mention HIF1AN knockout 293T cells that showed no detectable band compared to wild-type cells.

• Loading controls: Include appropriate loading controls such as GAPDH to normalize expression levels and ensure equal loading across samples .

• Treatment controls: For hypoxia-related studies, include both untreated and treated samples. For example, DFO (desferrioxamine) treatment is commonly used to mimic hypoxia, as shown in experiments with HIF-1 alpha detection .

• Secondary antibody-only control: Include a lane or sample with only secondary antibody to identify any non-specific binding.

• Isotype control: For immunocytochemistry or immunohistochemistry applications, include appropriate isotype controls matching the primary antibody's host species and isotype.

Including these controls in your experimental design will enhance data reliability and facilitate accurate interpretation of results when working with HIF1AN antibodies.

How can I study the interaction between HIF1AN and HIF-1 alpha in hypoxic conditions?

Studying the interaction between HIF1AN and HIF-1 alpha under hypoxic conditions requires careful experimental design. Based on the search results, consider the following methodological approach:

First, establish a reliable hypoxia model using chemical mimetics such as desferrioxamine (DFO). DFO treatment (1 mM overnight) has been shown to stabilize HIF-1 alpha in HepG2 and HeLa cells . This approach creates a controlled environment to study HIF1AN-mediated regulation of HIF-1 alpha without specialized hypoxia chambers.

For detecting both proteins simultaneously, use antibodies specifically validated for each target. For HIF-1 alpha, antibodies like MAB19351 have been validated to show increased expression after DFO treatment, with detection at approximately 120 kDa . For HIF1AN, antibodies described in the search results maintain consistent detection at 40-42 kDa regardless of oxygen conditions .

To study direct interactions, co-immunoprecipitation can be performed using either HIF1AN or HIF-1 alpha antibodies for pulldown, followed by Western blot detection of the binding partner. When conducting these experiments, it's essential to include input controls, IgG controls, and knockout validation where possible. For cellular localization studies, fluorescent immunocytochemistry can visualize the nuclear accumulation of HIF-1 alpha under hypoxic conditions while monitoring HIF1AN localization .

Alternative approaches include proximity ligation assays to visualize direct interactions in situ or CRISPR-based manipulation of either protein to assess functional consequences of the interaction.

What are the common troubleshooting issues with HIF1AN antibodies in Western blot applications?

When working with HIF1AN antibodies in Western blot applications, researchers may encounter several common technical issues. Based on the search results and general antibody troubleshooting principles, here are key challenges and solutions:

• Non-specific bands: Some HIF1AN antibodies may show non-specific interactions, particularly in automated systems. For example, the R&D Systems antibody MAB7874 showed non-specific interaction with the 230 kDa Simple Western standard . Solution: Optimize antibody concentration, increase washing steps, and use freshly prepared blocking buffers. Validate results using knockout controls when possible.

• Variable band intensity across tissue types: The search results show different expression levels of HIF1AN across tissues . Solution: Adjust loading amounts based on expected expression levels or use more sensitive detection methods for low-expressing samples.

• Optimization for different species: While many HIF1AN antibodies work across species (human, mouse, rat), optimization may be required for each species. Solution: Adjust antibody concentration based on the target species and verify cross-reactivity using positive controls from the species of interest.

• Buffer compatibility issues: Different immunoblot buffer systems may affect antibody performance. Solution: The search results mention Immunoblot Buffer Group 1 for certain applications . Follow manufacturer recommendations or test multiple buffer systems if encountering detection problems.

• Protein degradation: As an enzyme involved in post-translational modifications, HIF1AN may be sensitive to sample preparation conditions. Solution: Use fresh samples, add protease inhibitors during lysis, and keep samples cold throughout preparation.

For persistent issues, consider comparing results from multiple antibodies targeting different epitopes of HIF1AN to confirm findings and eliminate antibody-specific artifacts.

How can I assess HIF1AN activity rather than just protein expression?

Assessing HIF1AN enzymatic activity provides more functional information than simple protein expression analysis. Based on the search results and knowledge of HIF1AN function, consider the following methodological approaches:

• Hydroxylation activity assessment: HIF1AN functions by hydroxylating HIF-1 alpha at Asn-799 . To assess this activity, researchers can use antibodies that specifically recognize hydroxylated vs. non-hydroxylated forms of HIF-1 alpha. Alternatively, mass spectrometry can be employed to directly measure hydroxylation status of target residues.

• Functional readouts via HIF-1 transcriptional activity: Since HIF1AN inhibits HIF-1 transcriptional activity, measuring the expression of HIF-1 target genes (such as VEGF, GLUT1, or EPO) via qPCR or reporter assays can serve as an indirect measure of HIF1AN activity. Lower target gene expression under normoxic conditions indicates functional HIF1AN activity.

• Interaction-based activity assays: HIF1AN hydroxylation prevents interaction between HIF-1 alpha and transcriptional coactivators like p300 . Co-immunoprecipitation assays comparing the HIF-1/p300 interaction under various conditions can indirectly assess HIF1AN activity.

• Inhibitor-based approaches: Using known inhibitors of hydroxylases (such as DMOG or 2-OG analogs) can help determine if detected effects are specifically due to HIF1AN enzymatic activity.

• Oxygen-dependent enzymatic assays: Since HIF1AN is an oxygen sensor, comparing its activity under normoxic and hypoxic conditions provides functional information. This can be done using any of the above methods while manipulating oxygen levels.

When designing these experiments, include appropriate controls such as HIF1AN knockout/knockdown samples and oxygen level manipulations to establish the specificity of your activity assessments.

How can HIF1AN antibodies be used in cancer research studies?

HIF1AN antibodies have valuable applications in cancer research due to the critical role of hypoxia and oxygen sensing in tumor biology. Based on the search results, here are methodological approaches for using HIF1AN antibodies in cancer studies:

Immunohistochemical analysis of HIF1AN in tumor tissues can provide insights into oxygen sensing mechanisms within the tumor microenvironment. The search results indicate successful detection of HIF1AN in human lung cancer tissue using IHC with recommended dilutions of 1:50-1:500 . When performing these analyses, proper antigen retrieval is crucial, with TE buffer pH 9.0 or citrate buffer pH 6.0 recommended for optimal results .

For comparison of HIF1AN expression between tumor and normal tissues or across different cancer types, Western blot analysis using validated antibodies can quantify expression levels. The search results show successful detection of HIF1AN in glioblastoma (A172) cell lines , which can serve as positive controls for brain tumor studies.

To investigate the functional relationship between HIF1AN and hypoxia responses in cancer cells, researchers can use paired antibodies against both HIF1AN and HIF-1 alpha. Studies have demonstrated that DFO treatment (1 mM overnight) can be used to stabilize HIF-1 alpha in cancer cell lines like HepG2 and HeLa , allowing for assessment of how HIF1AN regulates the hypoxic response in these models.

Fluorescent immunocytochemistry can be employed to examine the subcellular localization of HIF1AN in cancer cells under varying oxygen conditions. The search results indicate that HIF1AN localizes to both cytoplasm and nuclei in human glioblastoma cells , providing a basis for studying localization-dependent functions in cancer biology.

What are the considerations for studying HIF1AN in developmental and stem cell research?

When investigating HIF1AN in developmental and stem cell contexts, several methodological considerations should be addressed based on the protein's diverse functions:

First, consider that HIF1AN has been shown to regulate NOTCH1 activity, which accelerates myogenic differentiation . This makes it particularly relevant for studies involving muscle development and stem cell differentiation into muscle lineages. When designing such experiments, C2C12 mouse myoblast cells can serve as an excellent model system, as the search results demonstrate successful detection of HIF1AN in these cells using both Western blot and fluorescent immunocytochemistry techniques .

Additionally, HIF1AN positively regulates ASB4 activity, which promotes vascular differentiation . This function makes HIF1AN antibodies valuable tools for studying angiogenesis and vascular development in embryonic or stem cell models. When investigating these processes, consider examining the relationship between HIF1AN, ASB4, and downstream vascular markers.

For developmental studies, it's important to note that HIF1AN can be detected in various mouse and rat tissues including brain, heart, skeletal muscle, hypothalamus, and colon , suggesting widespread expression during development. When analyzing developmental time points, adjust antibody concentrations appropriately as expression levels may vary throughout development.

In stem cell studies, the hydroxylation activity of HIF1AN may influence differentiation through multiple pathways beyond HIF-1 regulation. The search results indicate that HIF1AN hydroxylates specific residues in proteins involved in diverse cellular processes, including NFKB1, NFKBIA, and NOTCH1 . Consider examining these interactions when studying stem cell fate decisions.

For all developmental studies, knockout/knockdown validation becomes particularly important. The search results describe siRNA knockdown of HIF1AN in C2C12 cells and knockout 293T cell models , which provide templates for generating similar validation tools in developmental models.

How can I optimize immunofluorescence protocols for HIF1AN detection?

Optimizing immunofluorescence protocols for HIF1AN detection requires attention to several key experimental parameters. Based on the search results, the following methodological approach is recommended:

For cell fixation, immersion fixation has been successfully employed for both human and mouse cell lines . The specific fixation buffers aren't explicitly mentioned in the search results, but standard protocols typically use 4% paraformaldehyde for 10-15 minutes at room temperature for optimal antigen preservation while maintaining cellular architecture.

Antibody incubation parameters are critical for balancing signal strength and specificity. For human cell lines like A172 glioblastoma, the R&D Systems antibody MAB7874 worked effectively at 3 μg/mL when incubated for 3 hours at room temperature . For mouse C2C12 myoblast cells, a higher concentration of 25 μg/mL was required using the same antibody and incubation time , highlighting the importance of optimization for each cell type.

For secondary antibody detection, NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody has been validated for HIF1AN immunofluorescence . DAPI counterstaining effectively visualizes nuclei to assess HIF1AN subcellular localization .

When interpreting results, note that HIF1AN typically shows both cytoplasmic and nuclear localization in human cells like A172 , while in mouse C2C12 cells, staining is primarily localized to the cytoplasm . This differential localization may be biologically relevant or may require optimization of permeabilization conditions for complete epitope access.

For challenging samples, consider implementing Fluorescent ICC Staining protocols specifically designed for non-adherent cells as mentioned in the search results , which may provide better results for suspension cultures or weakly adherent cell populations.

How do different commercial HIF1AN antibodies compare in terms of applications and performance?

When selecting a HIF1AN antibody for research applications, understanding the comparative performance across commercial options is essential. Based on the search results, here is a methodological comparison of available antibodies:

For Western blot applications, all listed antibodies have been validated, but they differ in how they've been verified. The Bio-Rad antibody (VMA00490) stands out for validation using HIF1AN knockout 293T cells , providing strong evidence of specificity. The Abcam antibody (ab307829) has been validated using siRNA knockdown , another robust specificity verification method.

For immunohistochemistry applications, the Proteintech antibody (10646-1-AP) is the only one with explicit IHC validation in the search results, with recommended dilutions of 1:50-1:500 for human lung cancer tissue .

For immunofluorescence, the R&D Systems antibody (MAB7874) has been validated for both human and mouse cell lines, though with different optimal concentrations (3 μg/mL for human A172 cells vs. 25 μg/mL for mouse C2C12 cells) .

When selecting an antibody, consider your target species, application requirements, and preferred validation method. For multi-species studies, the Proteintech and R&D Systems antibodies offer the broadest validated reactivity across human, mouse, and rat samples.

How does detecting HIF1AN differ from detecting HIF-1 alpha in experimental settings?

Detecting HIF1AN differs significantly from detecting HIF-1 alpha in experimental settings due to their distinct regulation, expression patterns, and technical requirements. Understanding these differences is crucial for designing experiments involving the hypoxia pathway:

First, protein stability and expression conditions show marked differences. HIF-1 alpha is rapidly degraded under normoxic conditions and stabilized during hypoxia, requiring special handling for detection. The search results show that HIF-1 alpha becomes detectable in HepG2 and HeLa cells only after treatment with 1 mM DFO (desferrioxamine) overnight . In contrast, HIF1AN shows constitutive expression regardless of oxygen conditions, with consistent detection across various cell and tissue types without requiring hypoxia simulation .

The molecular weight for detection also differs substantially: HIF-1 alpha is detected at approximately 120 kDa , while HIF1AN appears at 40-42 kDa . This difference has implications for gel preparation, transfer conditions, and running times in Western blot applications.

Subcellular localization patterns also differ, affecting immunofluorescence protocol optimization. HIF-1 alpha shows predominantly nuclear localization after hypoxia induction , while HIF1AN shows both cytoplasmic and nuclear localization in human cells and primarily cytoplasmic localization in mouse cells .

For validation approaches, HIF-1 alpha antibodies are typically validated by comparing normoxic and hypoxic conditions, with hypoxia mimetics like DFO serving as positive controls . In contrast, HIF1AN antibodies are validated using knockout cells or siRNA knockdown , as their expression is not primarily oxygen-dependent.

When investigating both proteins simultaneously, experimental design should account for these differences, particularly when studying their interaction under varying oxygen conditions. The functional interaction between HIF1AN and HIF-1 alpha can be monitored by assessing HIF-1 alpha hydroxylation status or downstream target gene expression as indirect measures of HIF1AN activity.