ARNT2 Antibody

What is ARNT2 and why is it significant in research?

ARNT2 (Aryl Hydrocarbon Receptor Nuclear Translocator 2) is a member of the basic-helix-loop-helix-Per-Arnt-Sim (bHLH-PAS) superfamily of transcription factors that specifically recognizes the xenobiotic response element (XRE) . It plays crucial roles in normal glucose handling in pancreatic beta cell function in humans and mice, and serves as a dimeric partner for other PAS proteins such as HIF and SIM .

ARNT2 is primarily expressed in the central nervous system and developing kidney . Recent research has indicated that ARNT2 may be involved in improvement of cognitive impairment, suggesting potential therapeutic implications for neurological disorders . The protein forms heterodimers with hypoxia-inducible factor-1α (HIF-1α), serving a vital role in neuronal survival and cell proliferation .

What are the primary applications for ARNT2 antibodies in research?

ARNT2 antibodies are versatile tools used across multiple experimental applications:

For optimal results in IHC applications, antigen retrieval with TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 can be used as an alternative .

How should ARNT2 antibodies be stored and handled to maintain reactivity?

Proper storage and handling are crucial for maintaining antibody reactivity and preventing degradation:

Most ARNT2 antibodies should be stored at -20°C for long-term preservation . Many preparations contain glycerol and/or sodium azide as preservatives, allowing stability for up to one year when properly stored . Short-term storage at 2-8°C is acceptable for some formulations, but should be limited to reduce freeze-thaw cycles .

Working dilutions should be prepared fresh before use, and repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and loss of antibody activity. For antibodies stored in solution, aliquoting into single-use volumes is recommended to minimize freeze-thaw damage .

What controls should be included when using ARNT2 antibodies for the first time?

When validating ARNT2 antibodies for a specific application, several controls should be included:

• Positive Control: Use cell lines or tissues known to express ARNT2, such as HEK-293 cells, Jurkat cells, Raji cells, or brain tissue samples .

• Negative Control: Include samples with low or no ARNT2 expression, or use the primary antibody buffer without the antibody.

• Knockdown/Knockout Validation: If possible, use ARNT2 knockdown or knockout samples to confirm specificity. Published validations using this approach are available .

• Peptide Competition Assay: Pre-incubate the antibody with excess immunizing peptide to demonstrate binding specificity.

• Secondary Antibody Control: Include a sample treated only with secondary antibody to identify any non-specific binding.

For cellular localization studies, nuclear counterstaining (e.g., with DAPI) can help confirm the expected predominantly nuclear localization of ARNT2 .

How can I optimize Western blot protocols for detecting ARNT2?

Optimizing Western blot protocols for ARNT2 detection requires attention to several factors:

• Sample Preparation: Use fresh cell lysates prepared with protease inhibitors. For ARNT2 detection, HEK-293, Jurkat, and Raji cells have been validated as positive control sources .

• Protein Loading: Load 35-50 μg of total protein per lane for optimal detection .

• Gel Selection: Use 8-10% SDS-PAGE gels to properly resolve the 80-85 kDa ARNT2 protein .

• Transfer Conditions: For proteins of this size, transfer to nitrocellulose membranes using a wet transfer system at 100V for 60-90 minutes or 30V overnight at 4°C .

• Blocking: Block membranes with BLOTTO buffer supplemented with DL-histidine (20 mM) for 1 hour at room temperature .

• Antibody Incubation: Dilute primary antibody in blocking buffer (typically 1:500-1:1000) and incubate for 1 hour at room temperature or overnight at 4°C .

• Washing: Wash thoroughly with TTBS (Tris-buffered saline with Tween-20) for a total of 45 minutes with multiple buffer changes .

• Detection: Use enhanced chemiluminescence (ECL) and capture multiple exposures to identify the optimal signal .

For stronger signals, consider using HRP-conjugated secondary antibodies at 1:10,000 dilution and ensure a final PBS wash before detection to remove any residual Tween-20 .

How can I distinguish between ARNT and ARNT2 in experimental systems?

Distinguishing between ARNT (ARNT1) and ARNT2 requires careful approach since they share significant sequence homology (approximately 90% in the bHLH-PAS domains) :

• Antibody Selection: Use antibodies raised against regions where the proteins differ. Anti-ARNT2 antibodies targeting the N-terminal or central regions (amino acids 249-278) can provide specificity .

• Expression Patterns: ARNT is widely expressed in most tissues, while ARNT2 is predominantly expressed in the central nervous system, developing kidney, and specific cell types . This differential expression can help distinguish between them in tissue samples.

• Molecular Weight Discrimination: Though similar, ARNT2 (80-85 kDa) might migrate slightly differently than ARNT on SDS-PAGE gels .

• Functional Analysis: ARNT and ARNT2 have distinct functional properties. For example, ARNT2 has minimal ability to induce CYP1A1 expression compared to ARNT in response to TCDD treatment . These functional differences can be exploited in experimental designs.

• Genetic Approaches: Use specific siRNA or shRNA to selectively knockdown one protein while monitoring the other.

Research has shown that ARNT2 has limited capacity to replace ARNT in AHR-mediated signaling, suggesting functional differences despite their structural similarity . This can be used in functional discrimination assays.

What are the critical considerations for using ARNT2 antibodies in neurological research?

When studying ARNT2 in neurological contexts, several considerations are important:

• Tissue Fixation and Processing: For brain tissue, optimal fixation is crucial. Overfixation can mask epitopes, while underfixation may compromise tissue morphology. For ARNT2 detection in brain tissue, PFA fixation (4%, 20 min) followed by permeabilization with Triton X-100 (0.1%, 10 min) has been successful .

• Regional Expression Variations: ARNT2 expression varies across brain regions. Studies have shown significant expression in the hippocampus, particularly in CA1 and CA2 regions, which should be considered when designing experiments .

• Pathological State Considerations: ARNT2 levels change in response to pathological conditions. For example, ARNT2 has been shown to be upregulated in the brains of ischemic rats and may be involved in post-stroke depression (PSD) pathophysiology .

• Co-localization Studies: Consider co-staining with neuronal, glial, or vascular markers to determine the cellular distribution of ARNT2 in the nervous system. Nuclear counterstaining with DAPI (10 μg/ml, 10 min) helps confirm the primarily nuclear localization .

• Functional Pathway Analysis: In neurological research, consider ARNT2's role as a dimerization partner with HIF-1α and its involvement in hypoxic response, which is particularly relevant in ischemic conditions .

For behavioral studies, correlations between ARNT2 expression and cognitive parameters should be established, as ARNT2 has been suggested to be positively associated with improvement of cognitive impairment .

What are common issues with ARNT2 antibody staining and how can they be resolved?

When working with ARNT2 antibodies, researchers may encounter several challenges:

For immunofluorescence applications specifically, counterstaining actin with Alexa Fluor 555-conjugated Phalloidin (7 units/ml, 1h at 37°C) can help distinguish cellular compartments and improve interpretation of ARNT2 localization .

How should Western blot data for ARNT2 be interpreted, particularly regarding multiple bands?

Interpreting ARNT2 Western blot results requires careful analysis:

• Expected Molecular Weight: The calculated molecular weight of ARNT2 is 79 kDa (716 amino acids), but the observed molecular weight is typically 80-85 kDa . This discrepancy is common with transcription factors due to post-translational modifications.

• Multiple Bands: If multiple bands are observed:

  • Bands at 80-85 kDa represent full-length ARNT2

  • Lower molecular weight bands may indicate degradation products or isoforms

  • Higher molecular weight bands might represent post-translationally modified forms

• Tissue-Specific Variations: Expression patterns vary by tissue source. ARNT2 is most abundant in brain tissue, with expression also detected in cells derived from kidney, CNS, and retinal epithelium .

• Validation Approach: To confirm band specificity:

  • Compare with positive controls (HEK-293, Jurkat, or Raji cells)

  • Run parallel samples with ARNT2 knockdown/knockout

  • Use multiple antibodies targeting different epitopes

• Loading Controls: When comparing ARNT2 expression between samples, appropriate loading controls should be used. For nuclear proteins like ARNT2, lamin B or histone H3 may be more appropriate than typical cytoplasmic loading controls like GAPDH or β-actin.

It's important to note that ARNT2 detection may be influenced by experimental conditions affecting its dimerization with partners like AHR or HIF-1α, which can impact subcellular localization and potentially detection .

How can ARNT2 antibodies be applied in hypoxia and ischemia research?

ARNT2 antibodies are valuable tools in hypoxia and ischemia research due to ARNT2's role as a dimerization partner for HIF-1α:

• Hypoxic Response Monitoring: ARNT2 antibodies can be used to track changes in ARNT2 expression and localization under hypoxic conditions. Under hypoxia, HIF-1α alternatively binds with ARNT or ARNT2 (also designated as HIF-1β and HIF-2β), subsequently activating the hypoxic response element .

• Stroke Model Analysis: In ischemic stroke models, ARNT2 levels have been shown to be elevated. Antibodies can be used to quantify these changes and correlate them with functional outcomes . For immunohistochemical studies in ischemic brain tissue, blocking with 3% H₂O₂ followed by normal goat serum blocking has been effective .

• Therapeutic Target Validation: Since ARNT2 may be associated with improvement of cognitive impairment following ischemia, antibodies can help validate it as a potential therapeutic target .

• Comparative Analysis: Using antibodies against both ARNT and ARNT2 allows researchers to compare their relative contributions to the hypoxic response in different tissues or under different conditions .

• Co-immunoprecipitation Studies: ARNT2 antibodies can be used in co-IP experiments to identify interaction partners under hypoxic conditions, providing insights into regulatory mechanisms.

For ischemia studies, it's worth noting that ARNT2 has been specifically implicated in post-stroke depression (PSD), making it relevant for both acute and chronic post-ischemic processes .

What methodological approaches are recommended for studying ARNT2 in developmental contexts?

ARNT2 plays important roles in development, particularly in the central nervous system and kidney. When studying ARNT2 in developmental contexts:

• Developmental Timeline Sampling: Design experiments to capture ARNT2 expression across multiple developmental stages, as expression patterns change throughout development.

• Tissue Section Preparation: For embryonic or developmental tissues, proper fixation is crucial. For immunohistochemistry, 4% PFA fixation followed by careful sectioning at consistent thicknesses (typically 5-10 μm) is recommended .

• Co-localization Studies: Combine ARNT2 antibody staining with markers for developmental processes such as proliferation (Ki67), differentiation (lineage-specific markers), or apoptosis (TUNEL) to correlate ARNT2 expression with specific developmental events.

• Functional Studies: Complement antibody-based detection with functional studies:

  • Conditional knockout models

  • Temporal and spatial gene expression manipulation

  • Correlation with developmental phenotypes

• Interaction Partner Analysis: During development, ARNT2 functions through interactions with partners like SIM1 and HIF-1α. Co-immunoprecipitation using ARNT2 antibodies can help identify development-specific protein complexes.

• Quantification Approaches: For developmental studies, quantitative analysis is crucial:

  • Image analysis of staining intensity across developmental gradients

  • Western blot quantification normalized to appropriate loading controls

  • qPCR correlation with protein expression data

When interpreting developmental data, consider that ARNT2 expression is enriched in neural tissues and developing kidney, but expression patterns may change during different developmental stages and in response to environmental signals .

How do different epitope targets affect ARNT2 antibody performance in various applications?

The choice of epitope target significantly impacts ARNT2 antibody performance across applications:

The epitope target selection should be guided by the research question:

• For Isoform Discrimination: C-terminal antibodies may distinguish between potential splice variants.

• For Specificity vs. ARNT1: Antibodies targeting regions outside the highly conserved bHLH-PAS domains provide better discrimination between ARNT and ARNT2.

• For Functional Studies: Antibodies targeting functional domains may interfere with protein-protein interactions, which can be exploited in blocking studies but may complicate co-immunoprecipitation experiments.

• For Detecting Conformational Changes: Different epitope-targeting antibodies may have varying abilities to detect ARNT2 when it's engaged in protein complexes or under different cellular conditions.

When selecting an antibody, consider whether the immunogen was a short synthetic peptide or a larger fusion protein. Antibodies generated against fusion proteins (e.g., ARNT2 fusion protein Ag3536 ) may recognize conformational epitopes better than those raised against short peptides.

What are the implications of ARNT2 post-translational modifications for antibody detection?

Post-translational modifications (PTMs) of ARNT2 can significantly impact antibody recognition and experimental interpretation:

• Detection Considerations:

  • The calculated molecular weight of ARNT2 is 79 kDa, but it typically runs at 80-85 kDa on SDS-PAGE, suggesting the presence of PTMs .

  • Antibodies raised against unmodified peptides may have reduced affinity for modified ARNT2.

  • Some PTMs may mask epitopes, resulting in false-negative results in certain samples or conditions.

• Functional Implications:

  • ARNT2 function is regulated by PTMs, including phosphorylation, which can affect dimerization with partners like HIF-1α.

  • Antibodies specific to modified forms could help track ARNT2 activation status.

• Experimental Approaches:

  • To detect total ARNT2 regardless of modification state, use antibodies targeting regions less likely to be modified.

  • To study specific modifications, modification-specific antibodies may be required (though these are currently limited for ARNT2).

  • For comprehensive analysis, consider combining immunoprecipitation with mass spectrometry to identify PTMs.

• Sample Handling:

  • Include phosphatase inhibitors in lysis buffers when studying phosphorylated forms.

  • Consider native vs. denaturing conditions for immunoprecipitation, as some PTMs may affect protein folding and epitope accessibility.

• Data Interpretation:

  • Multiple bands on Western blots may represent differently modified forms rather than degradation products or non-specific binding.

  • Changes in apparent molecular weight under different cellular conditions may reflect changes in modification status rather than protein expression.

Understanding the potential PTMs of ARNT2 in your experimental system is crucial for proper interpretation of antibody-based detection results and may provide insights into regulatory mechanisms affecting ARNT2 function.