NUDT18 Antibody

What is NUDT18 and what is its primary function?

NUDT18 (nudix hydrolase 18), also known as MTH3, belongs to the Nudix hydrolase family that eliminates potentially toxic nucleotide metabolites from cells. It regulates the concentrations and availability of various nucleotide substrates, cofactors, and signaling molecules . NUDT18 is specifically active against 8-oxo-dGDP and hardly cleaves 8-oxo-dGTP. The protein has a molecular weight of approximately 36 kDa and consists of 323 amino acids . The enzyme plays a critical role in maintaining genome stability under oxidative stress by preventing the incorporation of damaged nucleotides into DNA.

What are the key applications for NUDT18 antibodies?

NUDT18 antibodies can be utilized in multiple experimental applications depending on research needs and antibody specifications:

It is recommended that researchers titrate each reagent in their specific testing system to obtain optimal results, as outcomes may be sample-dependent .

How should NUDT18 antibodies be stored for optimal performance?

NUDT18 antibodies should be stored at -20°C for long-term stability. They are typically stable for one year after shipment when stored properly. The common storage buffer is PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Aliquoting is generally unnecessary for -20°C storage, which simplifies handling. Some antibody preparations (20μl sizes) may contain 0.1% BSA as a stabilizer . When working with the antibody, avoid repeated freeze-thaw cycles to maintain optimal activity.

What species reactivity can be expected with NUDT18 antibodies?

The species reactivity of NUDT18 antibodies varies by product. From the search results, we can see the following patterns:

Researchers should select antibodies based on their target experimental model, with human reactivity being the most commonly available .

How does NUDT18 expression differ between cancerous and non-cancerous cells?

NUDT18 exhibits markedly elevated expression in noncancerous cell lines compared to their cancerous counterparts . This differential expression suggests potential roles in cancer biology that warrant further investigation. NUDT18-deficient cells have been reported to exhibit a noticeably reduced proliferation rate , suggesting that NUDT18 may be important for normal cell growth and division. The inverse relationship between NUDT18 expression and cancer status presents an interesting avenue for research into whether NUDT18 functions as a tumor suppressor or is downregulated as a consequence of cancerous transformation.

What is the relationship between NUDT18 and oxidative stress response?

As a member of the Nudix hydrolase family, NUDT18 plays a role in maintaining genome stability under oxidative stress . It specifically targets 8-oxo-dGDP, a product of oxidative damage to guanine nucleotides. By preventing the incorporation of oxidized nucleotides into DNA, NUDT18 helps prevent mutations and maintain genomic integrity. The specific activity against 8-oxo-dGDP but not 8-oxo-dGTP suggests a specialized role in the cellular response to oxidative damage that complements other Nudix family members like MTH1 and MTH2 . This functional specificity makes NUDT18 antibodies valuable tools for studying nucleotide pool sanitization mechanisms.

How can immunoprecipitation (IP) with NUDT18 antibodies be optimized for identifying protein-protein interactions?

To optimize immunoprecipitation with NUDT18 antibodies for protein-protein interaction studies:

• Lysate preparation: Use 1.0-3.0 mg of total protein lysate from cells known to express NUDT18 (e.g., THP-1 cells)

• Antibody amount: Use 0.5-4.0 μg of NUDT18 antibody, titrating to find optimal conditions

• Cross-linking considerations: For transient interactions, consider using chemical cross-linkers prior to cell lysis

• Wash stringency: Adjust salt and detergent concentrations in wash buffers to balance between specificity and sensitivity

• Controls: Include IgG control and input samples for comparison

• Analysis: Use mass spectrometry or Western blotting to identify co-immunoprecipitated proteins

For studying NUDT18's role in the DNA damage response pathway, mild lysis conditions may better preserve protein complexes. Positive IP has been detected in THP-1 cells, making them a good model system for optimization .

What are the critical validation steps for NUDT18 antibodies in various applications?

Proper validation of NUDT18 antibodies is essential for reliable research outcomes:

For immunohistochemistry and immunofluorescence applications, additional validation should include subcellular localization confirmation against known NUDT18 distribution patterns .

How can researchers troubleshoot weak or non-specific signals when using NUDT18 antibodies?

When troubleshooting weak or non-specific signals with NUDT18 antibodies:

• Sample preparation: Ensure proper cell lysis and protein extraction; NUDT18 is a 36 kDa protein that should be readily extractable with standard protocols

• Antibody concentration: Adjust dilution ratios (e.g., try 1:200 instead of 1:800 for IF/ICC) to find optimal signal-to-noise ratio

• Blocking optimization: Test different blocking agents (BSA, non-fat milk, serum) to reduce background

• Incubation conditions: Extend primary antibody incubation time (overnight at 4°C instead of 1 hour at room temperature)

• Detection systems: Try more sensitive detection methods (e.g., enhanced chemiluminescence for WB, tyramide signal amplification for IHC)

• Positive controls: Include known NUDT18-expressing cells like THP-1

• Epitope accessibility: Consider antigen retrieval methods for IHC or denaturation conditions for WB

• Antibody selection: Different clones target different regions of NUDT18 (N-terminal, C-terminal, or specific domains); switch antibodies if one region might be masked

What considerations should be made when using NUDT18 antibodies for studying its role in DNA damage responses?

When investigating NUDT18's role in DNA damage responses:

• Experimental timing: Establish appropriate time points post-DNA damage induction to capture dynamic changes in NUDT18 activity or localization

• Damage specificity: Different damage types (oxidative, UV, ionizing radiation) may affect NUDT18 differently due to its substrate specificity for 8-oxo-dGDP

• Cell type selection: Consider using both cancerous and non-cancerous cell lines given the differential expression patterns observed

• Co-localization studies: Combine NUDT18 antibodies with markers of DNA damage (γH2AX) or repair pathways to assess spatial relationships

• Functional assays: Complement antibody-based detection with enzymatic activity assays to correlate NUDT18 presence with function

• Stress induction methods: When studying oxidative stress responses, carefully calibrate H₂O₂ or other oxidant concentrations to avoid overwhelming cellular systems

• Genetic approaches: Consider using NUDT18 knockout/knockdown models alongside antibody studies to establish causality in observed phenotypes

How does NUDT18 (MTH3) function compare to other MTH family members?

NUDT18 (MTH3) functions as part of the MTH family alongside MTH1 and MTH2, all involved in sanitizing oxidized nucleotide pools:

NUDT18's specificity for 8-oxo-dGDP represents a specialized niche in protecting genome integrity, focusing on a different part of the nucleotide metabolism pathway than other MTH proteins . The distinct substrate preferences suggest non-redundant, complementary roles in the cellular response to oxidative stress. According to research cited in the search results, these proteins collectively maintain genome stability under oxidative stress conditions .

What protocols should be optimized when using NUDT18 antibodies for quantitative analysis of expression levels?

For quantitative analysis of NUDT18 expression levels:

• Sample normalization: Use housekeeping proteins appropriate for the experimental context and verify their stability under your conditions

• Standard curves: For ELISA applications, generate reliable standard curves using recombinant NUDT18 protein

• Loading control selection: When comparing cancerous vs. non-cancerous cells, select loading controls unaffected by cell transformation status

• Antibody saturation: Ensure working within the linear range of antibody detection to avoid saturation effects that could mask real differences

• Image analysis: For IF/ICC quantification, establish consistent exposure settings and analysis parameters across samples

• Reference standards: Include well-characterized cell lines with known NUDT18 expression levels (e.g., THP-1) as internal reference points

• Validation by orthogonal methods: Confirm antibody-based quantification with mRNA expression data or activity assays

• Statistical considerations: Apply appropriate statistical tests based on data distribution and experimental design

How can NUDT18 antibodies be effectively used in multiplexed imaging approaches?

For multiplexed imaging approaches with NUDT18 antibodies:

• Antibody selection: Choose NUDT18 antibodies raised in different host species than other target proteins to avoid cross-reactivity

• Fluorophore selection: Select fluorophores with minimal spectral overlap to reduce bleed-through

• Optimization strategy:

  • Begin with single-antibody staining to establish optimal dilutions (1:200-1:800 for IF/ICC)

  • Add antibodies sequentially, validating each step

  • Include appropriate controls for each antibody combination

• Sequential staining protocols: For targets requiring antibodies from the same species, consider sequential staining with intermediate blocking steps

• Sample preparation: Optimize fixation and permeabilization conditions that work for all target proteins

• Imaging parameters: Establish consistent acquisition settings for quantitative comparisons across experimental conditions

• Analysis considerations: Use appropriate computational tools for spectral unmixing and co-localization analysis

What novel research directions might NUDT18 antibodies enable in understanding cancer biology?

Novel research directions using NUDT18 antibodies in cancer biology:

• Biomarker development: Investigate NUDT18 as a potential diagnostic or prognostic marker based on its differential expression in cancerous versus non-cancerous cells

• Therapeutic targeting assessment: Use antibodies to monitor NUDT18 levels following treatment with compounds designed to modulate oxidative stress responses

• Cell cycle regulation studies: Explore the relationship between NUDT18 expression/localization and cell cycle progression, particularly given the reduced proliferation rate in NUDT18-deficient cells

• DNA damage response: Investigate NUDT18's role in cancer cells' response to chemotherapeutic agents that induce oxidative stress

• Tumor microenvironment: Examine how NUDT18 expression in cancer cells is affected by hypoxia, inflammation, or other microenvironmental factors

• Cancer stem cell biology: Compare NUDT18 levels between cancer stem cells and differentiated tumor cells to identify potential roles in stemness

• Resistance mechanisms: Study whether alterations in NUDT18 expression correlate with resistance to specific cancer therapies

• Personalized medicine applications: Determine if NUDT18 expression levels could predict response to particular treatment modalities

What are the recommended best practices for publishing research using NUDT18 antibodies?

When publishing research using NUDT18 antibodies, adhere to the following best practices:

• Antibody reporting: Provide complete information including catalog number (e.g., ABIN7249196), clone (for monoclonals), host species, and target epitope information

• Validation documentation: Include details of antibody validation performed specifically for your experimental context

• Reproducibility considerations: Report number of experimental replicates and statistical approaches

• Methodological transparency: Detail antibody dilutions, incubation conditions, detection methods, and image acquisition parameters

• Controls description: Clearly describe all positive and negative controls used to validate findings

• Limitations acknowledgment: Discuss any limitations related to antibody cross-reactivity or specificity

• Data availability: Consider sharing original unprocessed images or other raw data

• Citation practice: Cite the relevant publications that characterize NUDT18's function (e.g., its role in genome stability under oxidative stress)