NTHL1 Antibody

What is NTHL1 and what cellular functions does it perform?

NTHL1, also known as Endonuclease III-like protein 1 or OCTS3, is a 312 amino acid protein belonging to the Nth/MutY family. It functions as a bifunctional DNA glycosylase/AP lyase that initiates repair of oxidatively damaged pyrimidines through the base excision repair (BER) pathway. NTHL1 localizes in the nucleus and is widely expressed throughout the body, with highest expression levels in heart tissue and lowest levels in lung and liver . It possesses both apurinic/apyrimidinic endonuclease activity and DNA N-glycosylase activity, allowing it to incise damaged DNA at cytosines, thymines, and guanines. The protein acts on damaged DNA strands 5' from the damaged site and plays a critical role in repairing both oxidative DNA damage and spontaneous mutagenic lesions .

What types of NTHL1 antibodies are available for research applications?

Multiple NTHL1 antibodies are available for research applications, with variations in the epitope targets, host species, and applications. Most commonly, researchers utilize rabbit polyclonal antibodies against NTHL1, though monoclonal options also exist. These antibodies target different amino acid regions of the NTHL1 protein, including:

• Central region antibodies (AA 88-117)

• Full-length antibodies (AA 31-312)

• N-terminal region antibodies (AA 1-280)

The selection of an antibody depends on the specific research application, with most NTHL1 antibodies being validated for Western blotting, while some are also suitable for immunoprecipitation, ELISA, immunohistochemistry, and immunofluorescence applications .

How should NTHL1 antibodies be stored to maintain optimal activity?

NTHL1 antibodies require specific storage conditions to maintain their functionality and prevent degradation. Based on manufacturer guidelines, NTHL1 antibodies should be stored at -20°C, where they remain stable for one year after shipment . The typical storage buffer consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which helps prevent microbial growth and maintain protein stability . For smaller quantities (20μl sizes), the formulation may contain 0.1% BSA as a stabilizer. Importantly, aliquoting is generally unnecessary for -20°C storage, which simplifies lab handling protocols. When working with the antibody, avoid repeated freeze-thaw cycles which can compromise antibody integrity and binding efficiency .

What are the optimal dilutions for using NTHL1 antibody in Western blot experiments?

For optimal Western blot results with NTHL1 antibody, consider the following protocol recommendations:

How can researchers validate the specificity of NTHL1 antibodies?

Validating antibody specificity is critical for ensuring reliable research outcomes. For NTHL1 antibodies, several complementary approaches should be employed:

• Positive and negative control samples: Compare expression in cells known to express NTHL1 (HEK-293, HeLa, and K-562 cells) versus cells with lower or no expression .

• Knockdown validation: Perform siRNA or shRNA-mediated knockdown of NTHL1 to confirm signal reduction in Western blot analysis.

• Overexpression studies: Generate cells stably expressing HA-tagged NTHL1 using lentiviral constructs, as described in published protocols . This approach allows antibody validation against artificially elevated levels of the target protein.

• Blocking peptide competition: Pre-incubate the antibody with the immunizing peptide (from the central region AA 88-117) to demonstrate signal reduction .

• Cross-reactivity testing: Evaluate potential cross-reactivity by testing the antibody against human, mouse, and rat samples, as NTHL1 antibodies have demonstrated reactivity across these species .

This multi-faceted validation approach ensures that the observed signal is specific to NTHL1 and not due to non-specific binding or cross-reactivity with other proteins.

What methodological considerations are important when using NTHL1 antibody for immunoprecipitation experiments?

When performing immunoprecipitation (IP) with NTHL1 antibodies, several methodological considerations are critical for successful experiments:

• Cell lysis conditions: For optimal NTHL1 immunoprecipitation, cells should be lysed using a buffer containing 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 5% glycerol, supplemented with protease and phosphatase inhibitors .

• Lysis duration: Maintain cells on ice during lysis for approximately 40 minutes with intermittent vortexing to ensure complete protein extraction while preserving protein-protein interactions .

• Nucleic acid interference: To determine whether NTHL1 interactions with other proteins (such as XPG) are DNA or RNA-mediated, pretreat lysates with ethidium bromide (10 μg/ml), RNase A (20 μg/ml), or DNase (100 units/ml) for 30 minutes on ice before IP .

• Tag-based IP approaches: For tagged NTHL1 constructs, commercial kits like the Pierce HA Tag IP/Co-IP Kit provide standardized protocols for effective immunoprecipitation .

• Elution conditions: Carefully optimize elution conditions to maintain protein structure and interaction integrity for downstream applications like Western blot analysis or mass spectrometry.

• Controls: Always include appropriate negative controls (IgG, empty vector) to identify non-specific binding patterns.

These methodological considerations help ensure specific isolation of NTHL1 protein complexes while preserving their biological interactions for accurate characterization.

How does NTHL1 expression impact cellular sensitivity to chemotherapeutic agents?

Recent research has revealed a significant relationship between NTHL1 expression levels and cellular sensitivity to platinum-based chemotherapeutic agents, particularly cisplatin. Studies investigating non-small cell lung carcinoma (NSCLC) cell lines demonstrated that the relationship between NTHL1 expression and cisplatin sensitivity follows a consistent pattern .

Cell lines with lower NTHL1 expression, such as the H522 NSCLC line, exhibit higher resistance to cisplatin treatment. Conversely, when NTHL1 is overexpressed in these resistant cells, they become more sensitive to cisplatin-induced cytotoxicity . This suggests that NTHL1 expression levels may serve as a potential biomarker for predicting cisplatin response in certain cancers.

The mechanism underlying this sensitivity pattern appears to involve interactions between NTHL1 and nucleotide excision repair (NER) proteins, particularly XPG. This interaction may create a situation where NTHL1 overexpression paradoxically interferes with effective DNA repair processes by disrupting the coordinated activities of different DNA repair pathways .

These findings have significant implications for cancer treatment strategies, suggesting that:

• NTHL1 expression testing might help identify patients likely to respond to cisplatin therapy

• Targeted modulation of NTHL1 could potentially sensitize resistant tumors to platinum-based treatments

• The interaction between different DNA repair pathways may create unexploited vulnerabilities in cancer cells

What is known about the interaction between NTHL1 and other DNA repair proteins?

NTHL1 functions within a complex network of DNA repair proteins, with particularly important interactions with nucleotide excision repair (NER) pathway components. Research using co-immunoprecipitation and mass spectrometry approaches has identified several key protein-protein interactions:

• NTHL1-XPG interaction: NTHL1 demonstrates a significant physical interaction with XPG, a critical endonuclease in the NER pathway . This interaction appears to impact cellular sensitivity to DNA damaging agents, particularly cisplatin.

• DNA-independent interaction: Experimental evidence suggests that the NTHL1-XPG interaction occurs independently of DNA bridging, as treatment with ethidium bromide or DNase does not disrupt the interaction .

• Functional consequences: Overexpression of NTHL1 can interfere with normal DNA repair processes, potentially by disrupting the coordinated activities of the base excision repair (BER) and NER pathways.

The interaction between NTHL1 and other DNA repair proteins appears to play a critical role in determining cellular responses to genotoxic stress, with implications for both normal genomic maintenance and cancer treatment responses.

What role does NTHL1 subcellular localization play in its biological function?

Research using immunofluorescence microscopy with tagged NTHL1 constructs has demonstrated that while the majority of NTHL1 protein is found in the nucleus under normal conditions, its distribution pattern can change in response to:

• Oxidative stress: Increased oxidative damage may alter NTHL1 distribution within nuclear subcompartments to coordinate with other DNA repair factors

• Cell cycle phase: NTHL1 localization patterns may shift during different cell cycle phases, with potential implications for repair activity timing

• Protein overexpression: When artificially overexpressed, NTHL1 distribution patterns may change, potentially contributing to the altered cellular phenotypes observed in overexpression studies

The regulated nuclear localization of NTHL1 is critical for its proper function in DNA repair pathways. Changes in this localization pattern, whether through mutation, overexpression, or cellular stress, may contribute to pathological states including genomic instability and cancer development.

What are common issues when detecting NTHL1 in Western blot experiments and how can they be resolved?

When working with NTHL1 antibodies in Western blot applications, researchers may encounter several common challenges. Here are the most frequently reported issues and their solutions:

• Weak or absent signal:

  • Increase antibody concentration within the recommended range (1:1000-1:4000)

  • Extend primary antibody incubation time to overnight at 4°C

  • Increase protein loading (40-60 μg per lane)

  • Use enhanced chemiluminescence (ECL) detection systems with higher sensitivity

• Multiple bands or high background:

  • Optimize blocking conditions (try 5% BSA instead of milk for blocking)

  • Increase washing duration and frequency (4-5 washes for 10 minutes each)

  • Decrease antibody concentration

  • Pre-absorb the antibody with cell/tissue lysate from a species different from your target

• Inconsistent results between experiments:

  • Standardize protein extraction protocols

  • Use fresh samples and avoid repeated freeze-thaw cycles

  • Maintain consistent transfer conditions

  • Include loading controls and positive control samples

• Incorrect molecular weight detection:

  • NTHL1 should appear at approximately 34 kDa

  • If bands appear at different sizes, consider post-translational modifications, degradation products, or isoforms

  • Optimize gel percentage (10-12% recommended for NTHL1)

Implementing these troubleshooting strategies can significantly improve the reliability and consistency of NTHL1 detection in Western blot experiments.

How can researchers optimize NTHL1 antibody-based immunofluorescence experiments?

Optimizing immunofluorescence (IF) experiments with NTHL1 antibodies requires attention to several critical parameters:

• Fixation method: Compare paraformaldehyde (4%, 10-15 minutes) versus methanol fixation (ice-cold, 10 minutes) to determine which better preserves NTHL1 epitopes while maintaining cellular architecture.

• Permeabilization: Since NTHL1 is a nuclear protein , ensure adequate nuclear permeabilization using 0.1-0.5% Triton X-100 in PBS for 10 minutes at room temperature.

• Antigen retrieval: For formalin-fixed samples, heat-induced epitope retrieval in citrate buffer (pH 6.0) may improve antibody accessibility to nuclear NTHL1.

• Antibody dilution optimization: Start with a dilution range of 1:100-1:500 for primary antibody and optimize based on signal-to-noise ratio.

• Signal amplification: Consider using tyramide signal amplification or biotinylated secondary antibodies with fluorescent streptavidin for weak signals.

• Controls: Always include:

  • Positive control (cells known to express NTHL1, such as HEK-293 or HeLa cells)

  • Negative control (primary antibody omission)

  • Nuclear counterstain (DAPI or Hoechst) to confirm nuclear localization

• Co-localization studies: When investigating NTHL1 interactions with other proteins (such as XPG) , carefully select fluorophores with minimal spectral overlap and use appropriate microscopy techniques (confocal microscopy recommended).

Following these optimization strategies will help ensure specific detection of NTHL1 in immunofluorescence applications with minimal background and maximum signal specificity.

How is NTHL1 antibody research contributing to our understanding of cancer development?

NTHL1 antibody-based research has provided critical insights into the relationship between DNA repair defects and cancer development. Recent studies utilizing NTHL1 antibodies have revealed several important findings:

• NTHL1 as a tumor suppressor: Immunohistochemical analysis using NTHL1 antibodies has demonstrated altered expression patterns in various cancer types, supporting its role as a tumor suppressor gene.

• Overexpression paradox: Contrary to expectations, research has shown that NTHL1 overexpression can lead to the acquisition of cancer hallmarks in non-tumorigenic immortalized cells . This paradoxical finding suggests that precise regulation of NTHL1 levels is critical for normal cellular function.

• Interaction with NER proteins: Immunoprecipitation studies using NTHL1 antibodies have identified interactions with nucleotide excision repair (NER) proteins, particularly XPG . This crosstalk between repair pathways appears to influence genomic stability and cancer susceptibility.

• Biomarker potential: Differential expression of NTHL1 in cancer tissues, as detected by antibody-based methods, may serve as a potential biomarker for treatment response prediction, particularly for platinum-based chemotherapies .

These findings highlight the complex role of NTHL1 in cancer biology and suggest that further antibody-based research could lead to new diagnostic and therapeutic approaches targeting DNA repair mechanisms in cancer.

What methodological advances are improving the study of NTHL1 protein interactions?

Recent methodological advances have significantly enhanced our ability to study NTHL1 protein interactions, providing deeper insights into its functional networks:

• Advanced proximity labeling techniques: Methods like BioID and APEX2 proximity labeling are being applied to NTHL1 research, allowing identification of transient or weak interactions that might be missed by traditional co-immunoprecipitation approaches.

• Live-cell imaging with fluorescently tagged NTHL1: Development of stable cell lines expressing fluorescently tagged NTHL1 enables real-time monitoring of protein dynamics and interactions in response to DNA damage .

• Sequential deletion mutants: Creation of sequential 20-amino-acid deletion mutants of NTHL1 has enabled mapping of specific protein interaction domains . This approach helps identify critical regions for protein-protein interactions and functional activities.

• Controlled expression systems: Tetracycline-inducible expression systems for NTHL1, as described in recent publications, allow precise control over expression levels to study dosage effects on cellular phenotypes .

• Mass spectrometry-based interactome analysis: Application of high-resolution mass spectrometry to analyze NTHL1 immunoprecipitates has expanded our understanding of its protein interaction network .

These methodological advances are providing unprecedented insights into NTHL1 biology and its role in DNA repair networks, with potential implications for understanding disease processes and developing novel therapeutic approaches.