nudt17 Antibody

What is NUDT17 and what cellular functions does it perform?

NUDT17 (Nudix Nucleoside Diphosphate Linked Moiety X-Type Motif 17) belongs to the NUDIX hydrolase family of enzymes that are involved in cellular metabolism, homeostasis, and mRNA processing. These enzymes are highly conserved throughout all domains of life . NUDT17's specific function remains somewhat unclear, as comprehensive biochemical screenings have failed to identify clear substrates for this enzyme, suggesting that it may require different conditions than those typically explored in standard assays . Unlike some other NUDIX family members that have well-characterized substrates and functions, NUDT17 represents one of several members whose physiological roles require further investigation.

While the exact function remains to be fully elucidated, the NUDIX family generally catalyzes the hydrolysis of nucleoside diphosphates linked to other moieties, and different members have varying substrate specificities. Research into NUDT17's role in cellular processes continues, with particular interest in potential connections to stress responses and cancer biology, as other NUDIX family members have shown relevance in these areas .

How should NUDT17 antibody specificity be validated?

Validating NUDT17 antibody specificity requires a multi-faceted approach to ensure reliable experimental results:

• Western blot with positive and negative controls: Run samples from tissues/cells known to express NUDT17 alongside those with confirmed absence or knockdown of NUDT17. Compare band patterns and molecular weights, expecting a primary band at the predicted molecular weight of NUDT17 .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide (such as the synthetic peptide corresponding to AA 168-217 of human NUDT17) before application to your sample . Specific binding should be blocked, resulting in signal reduction.

• Knockout/knockdown verification: Test the antibody on samples from NUDT17 knockout models or cells treated with siRNA targeting NUDT17. The specific band should disappear or be significantly reduced.

• Cross-reactivity assessment: Test the antibody against recombinant proteins of closely related NUDIX family members to ensure it does not cross-react with similar proteins, particularly important given the conserved domains within this enzyme family .

• Immunoprecipitation followed by mass spectrometry: This can confirm that the antibody is pulling down NUDT17 specifically rather than other proteins.

The validation approach should be matched to your specific application (Western blot, ELISA, immunohistochemistry, etc.) as antibody performance can vary between techniques.

What are optimal storage conditions for maintaining NUDT17 antibody activity?

To maintain NUDT17 antibody activity over time, follow these evidence-based storage practices:

• Short-term storage: Store at 4°C for periods of days to weeks, ensuring the antibody remains in the buffer recommended by the manufacturer .

• Long-term storage: Store at -20°C, as recommended for most antibody preparations including NUDT17 antibodies .

• Aliquoting: Upon receipt, divide the antibody into small working aliquots before freezing to avoid repeated freeze-thaw cycles, which can significantly degrade antibody quality.

• Reconstitution: For lyophilized NUDT17 antibodies, add the recommended volume of distilled water (typically 50 μL) to achieve the standard concentration of 1 mg/mL .

• Buffer conditions: NUDT17 antibodies are often lyophilized in PBS buffer with 2% sucrose for stability . Maintain these conditions when storing reconstituted antibody.

• Avoid freeze-thaw cycles: Each freeze-thaw cycle can reduce antibody activity by 5-20%. Limit these cycles to fewer than 5 total for any aliquot .

• Contamination prevention: Use sterile technique when handling antibodies to prevent microbial growth.

Monitor antibody performance periodically with positive controls to ensure continued activity, especially when using antibodies that have been stored for extended periods.

How can NUDT17 antibodies be used to investigate potential substrates of this enzyme?

• Immunoprecipitation-coupled enzyme assays: Use NUDT17 antibodies to immunoprecipitate the native enzyme from cellular extracts, then perform activity assays with candidate substrates. This approach preserves potential cofactors or post-translational modifications required for activity.

• Substrate-trapping mutants: Generate catalytically inactive NUDT17 variants that can still bind but not hydrolyze substrates. Use antibodies to immunoprecipitate these mutants and identify trapped substrates by mass spectrometry.

• Proximity labeling: Combine NUDT17 antibodies with proximity labeling approaches (BioID or APEX) to identify proteins and metabolites that interact with NUDT17 in cellular contexts, potentially revealing substrate candidates.

• Comparative metabolomics: Compare metabolite profiles between wild-type cells and NUDT17-depleted cells (using siRNA approaches as demonstrated with other NUDIX hydrolases ). Use NUDT17 antibodies to confirm knockdown efficiency in parallel experiments.

• In vitro reconstitution systems: Purify NUDT17 using antibody-based affinity columns and test against panels of candidate substrates based on known preferences of related NUDIX enzymes, particularly focusing on modified nucleotides .

This systematic approach is crucial since NUDT17 substrate identification has proven challenging in conventional high-throughput biochemical screens that successfully identified substrates for other NUDIX family members .

How does NUDT17 expression change in cancer cells compared to normal tissues?

The expression profile of NUDT17 across normal and cancer tissues presents a complex picture that requires careful investigation:

• Expression databases analysis: Analyze TCGA (The Cancer Genome Atlas) and HPA (Human Protein Atlas) data for NUDT17 expression patterns across tissue types. Unlike some NUDIX family members like NUDT1, which is significantly overexpressed in most cancers, or NUDT4, which is consistently downregulated, NUDT17 shows more variable expression patterns .

• Cancer-specific expression changes: Examine NUDT17 expression using antibody-based methods such as immunohistochemistry and Western blotting in paired normal-tumor samples. NUDIX family members form distinct clusters of expression in cancers, and understanding where NUDT17 fits provides insight into its potential role .

• Co-expression analysis: Investigate genes co-expressed with NUDT17, which may reveal functional relationships and potential involvement in cancer-related pathways. Hierarchical clustering analysis can position NUDT17 relative to other NUDIX enzymes with known cancer associations .

• Functional impact assessment: While some NUDIX enzymes like NUDT1 and NUDT2 significantly impact cancer cell viability upon depletion, NUDT17's effect should be similarly assessed using siRNA knockdown followed by antibody confirmation of depletion efficiency .

• Cell cycle effects: Analyze whether NUDT17 depletion affects cell cycle distribution using DNA content approaches, as other NUDIX enzymes show specific cell cycle perturbations in cancer cells .

The analysis should account for tissue-specific expression patterns, as NUDIX enzyme expression varies significantly across tissue types in both normal and cancer samples .

What methodological approaches can investigate functional redundancy between NUDT17 and other NUDIX hydrolases?

Investigating functional redundancy among NUDIX family members requires sophisticated experimental designs:

• Pairwise knockdown studies: Conduct systematic double siRNA-mediated knockdowns of NUDT17 paired with each other NUDIX hydrolase, following the approach demonstrated for comprehensive NUDIX family analysis . Measure viability and cell cycle effects to identify synthetic lethal or rescue relationships.

• Multiplicative model analysis: Apply the multiplicative model of genetic interactions to determine whether double knockdowns produce aggravating, nonsignificant, or alleviating effects compared to single knockdowns .

• Substrate competition assays: In vitro, test whether purified NUDT17 and other NUDIX enzymes compete for the same substrates using antibody-purified enzymes and biochemical assays.

• Structural and sequence analysis: Use bioinformatics to identify structural similarities in catalytic domains that might suggest functional overlap. Confirm predictions with biochemical assays using antibody-validated proteins.

• Rescue experiments: Deplete NUDT17 and assess whether overexpression of other NUDIX enzymes can rescue the phenotype, indicating functional redundancy.

• Domain swapping: Create chimeric proteins between NUDT17 and other NUDIX hydrolases to map functional domains, then use antibodies to confirm expression and localization of these constructs.

This systematic approach is particularly important since functional redundancy may explain why individual knockdowns of some NUDIX enzymes, including NUDT17, produce limited phenotypes in certain contexts .

What are the optimal conditions for using NUDT17 antibodies in Western blotting?

For optimal Western blotting results with NUDT17 antibodies, follow these methodologically rigorous protocols:

• Antibody concentration: Use NUDT17 antibody at 0.5 μg/mL for Western blotting applications .

• Secondary antibody dilution: HRP-conjugated secondary antibody should be diluted 1:50,000-100,000 for optimal signal-to-noise ratio .

• Sample preparation: Extract proteins using RIPA buffer supplemented with protease inhibitors. Denature samples at 95°C for 5 minutes in Laemmli buffer with β-mercaptoethanol.

• Gel percentage: Use 10-12% SDS-PAGE gels for optimal resolution of NUDT17, which has a molecular weight in the 20-25 kDa range.

• Transfer conditions: Transfer proteins to PVDF membranes at 100V for 1 hour in ice-cold transfer buffer containing 20% methanol, or at 30V overnight at 4°C for improved transfer efficiency.

• Blocking conditions: Block membranes with 5% non-fat dry milk in TBST for 1 hour at room temperature to minimize non-specific binding.

• Antibody incubation: Incubate with primary NUDT17 antibody overnight at 4°C in blocking buffer, followed by three 10-minute washes with TBST.

• Detection method: Use enhanced chemiluminescence (ECL) for detection, with exposure times optimized based on signal strength.

• Positive controls: Include purified recombinant NUDT17 protein or lysates from cells known to express NUDT17 as positive controls.

• Stripping and reprobing: If needed, use mild stripping buffer (200 mM glycine, 0.1% SDS, 1% Tween 20, pH 2.2) for 10 minutes at room temperature, followed by blocking and reprobing with different antibodies.

How can researchers troubleshoot non-specific binding when using NUDT17 antibodies?

When encountering non-specific binding with NUDT17 antibodies, implement these troubleshooting strategies:

• Titrate antibody concentration: Test serial dilutions of the NUDT17 antibody beyond the recommended 0.5 μg/mL for Western blotting to identify the optimal concentration that maximizes specific signal while minimizing background.

• Optimize blocking conditions: Compare different blocking agents (BSA, casein, commercial blocking buffers) at various concentrations (3-5%) and durations (1-3 hours) to identify conditions that most effectively reduce non-specific binding.

• Increase washing stringency: Extend TBST washing steps (3-5 washes of 10-15 minutes each) and consider adding higher concentrations of Tween-20 (0.1-0.3%) to washing buffers.

• Validate with peptide competition: Pre-incubate the antibody with the immunizing peptide before application. Specific bands should disappear, while non-specific bands will remain .

• Cross-adsorption: Pre-incubate the antibody with lysates from cells not expressing NUDT17 to remove antibodies that bind to non-target proteins.

• Alternative extraction methods: Try different lysis buffers (NP-40, CHAPS, Triton X-100 based) as some may better preserve epitope recognition while reducing non-specific interactions.

• Sample preparation modification: Optimize sample denaturation conditions (temperature, time, reducing agent concentration) to ensure complete protein unfolding without epitope destruction.

• Consider membrane type: Compare PVDF and nitrocellulose membranes, as binding properties differ and can affect background levels.

• Secondary antibody optimization: Test different sources and dilutions of secondary antibodies (1:50,000-100,000 range recommended for HRP-conjugated antibodies with NUDT17) .

• Use of FPLC-purified antibody fractions: For persistent non-specific binding issues, consider using affinity-purified antibody fractions.

What normalization methods are appropriate when analyzing NUDT17 antibody reactivity in protein microarrays?

When analyzing NUDT17 antibody reactivity in protein microarrays, employ these methodologically sound normalization approaches:

• Control-based initial normalization: Normalize array data using the mean values obtained from IgG controls and standard fluorescence markers on all arrays to account for technical variations in detection .

• Replicate array averaging: Average adjusted values for each antigen across replicate arrays to reduce random variation and increase reliability of measurements .

• Interquartile normalization: Express values as a percentage of interquartile differences per array using the formula:
  $\frac{(Observed\;value)-(25th\;percentile)}{(75th\;percentile)-(25th\;percentile)}$

• Quantile normalization: Apply standard quantile normalization to ensure that all arrays have identical median and quartile values, allowing for valid interarray comparisons .

• Baseline reactivity determination: Use the lower quartile (25th percentile) of all values obtained for NUDT17 with all sera as the baseline reactivity .

• Positive reaction threshold: Define positive reactivity as values exceeding 2.5× the interquartile difference above the 75th percentile, which provides a stringent cutoff that accounts for the distribution of reactivity across samples .

• Quality control metrics: Validate normalization by ensuring that standard markers (e.g., Cy3-biotin-BSA and IgG/IgM dilutions) show reproducibility within quadruplicates with a target CV of approximately 5.6% .

• Background subtraction: Subtract values from buffer-only spots to eliminate instrument background noise.

• Secondary antibody reactivity adjustment: Adjust all array values within each array to account for differences in secondary antibody detection efficiency .

• BCCP fusion tag consideration: When comparing antigen reactivity across different sera, consider adjusting for potential reactivity to the BCCP tag that enables attachment of proteins to the slide surface .

These normalization methods ensure robust analysis of NUDT17 antibody reactivity while minimizing technical variation and allowing for valid comparisons across different experimental conditions and sample types.

How can NUDT17 antibodies be effectively used in immunohistochemistry applications?

For optimal immunohistochemistry (IHC) results with NUDT17 antibodies, implement these methodological strategies:

• Fixation optimization: Compare formalin-fixed paraffin-embedded (FFPE) samples with frozen sections to determine which better preserves NUDT17 epitopes. For FFPE tissues, optimize fixation time (12-24 hours) to balance structural preservation with antigen accessibility.

• Antigen retrieval method selection: Test multiple antigen retrieval methods (heat-induced epitope retrieval in citrate buffer pH 6.0, EDTA buffer pH 9.0, or enzymatic retrieval with proteinase K) to determine which best exposes NUDT17 epitopes without destroying tissue morphology.

• Blocking protocol: Implement a dual blocking approach using 3-5% normal serum matching the secondary antibody host species, followed by an avidin-biotin blocking step if using biotinylated detection systems.

• Primary antibody titration: Test serial dilutions of the NUDT17 antibody, starting from the recommended concentration for Western blotting (0.5 μg/mL) and adjusting based on signal-to-noise ratio.

• Incubation conditions: Compare overnight incubation at 4°C with shorter incubations (1-3 hours) at room temperature to determine optimal binding conditions.

• Detection system selection: Compare sensitivity and specificity of different detection systems (polymer-based, tyramide signal amplification, or conventional avidin-biotin complex) for NUDT17 visualization.

• Counterstain compatibility: Select appropriate counterstains (hematoxylin for nuclear visualization, methyl green for better contrast with DAB) based on NUDT17's cellular localization.

• Multi-labeling approach: For co-localization studies, use sequential immunostaining with antibodies raised in different host species or implement tyramide-based multiplexing.

• Positive and negative controls: Include tissues known to express NUDT17 as positive controls, while using isotype control antibodies and NUDT17-depleted tissues as negative controls.

• Automated quantification: Implement digital image analysis using software platforms that can distinguish cellular compartments for accurate quantification of NUDT17 expression patterns.

This comprehensive approach ensures reliable detection and characterization of NUDT17 expression in tissue samples while minimizing artifacts and background staining.

What approaches can determine NUDT17 subcellular localization?

Determining NUDT17's precise subcellular localization requires a multi-dimensional methodological approach:

• Immunofluorescence microscopy: Use NUDT17 antibodies for immunofluorescence staining combined with confocal microscopy for high-resolution localization. Co-stain with markers for specific organelles (DAPI for nucleus, MitoTracker for mitochondria, ER-Tracker for endoplasmic reticulum).

• Subcellular fractionation with Western blotting: Separate cellular components (nucleus, cytoplasm, mitochondria, ER, etc.) using differential centrifugation techniques, then probe fractions with NUDT17 antibodies via Western blotting to identify enrichment in specific compartments.

• Proximity ligation assay (PLA): Combine NUDT17 antibodies with antibodies against known compartment-specific proteins to visualize proximity (<40 nm) through fluorescent signal generation only when proteins are closely associated.

• Electron microscopy immunogold labeling: For highest resolution localization, use gold-conjugated secondary antibodies against NUDT17 primary antibodies for visualization in transmission electron microscopy.

• Live-cell imaging: Create fluorescent protein fusions with NUDT17 and validate localization patterns using antibodies against endogenous NUDT17 to confirm that fusion constructs maintain native localization.

• Stimulus-dependent relocalization: Investigate whether cellular stresses (oxidative stress, nutrient deprivation, etc.) cause NUDT17 to relocalize, as other NUDIX family members respond to cellular stress conditions .

• Pharmacological interventions: Use drugs that disrupt specific organelles or cellular structures and assess effects on NUDT17 localization to identify dynamic relationships with cellular compartments.

• Deletion construct analysis: Create truncation mutants of NUDT17 to identify localization signals within the protein sequence, validating with antibodies that recognize different epitopes.

• Bioinformatic prediction validation: Test computational predictions of localization signals within NUDT17 using antibodies against the endogenous protein to confirm actual cellular distribution.

This comprehensive approach provides convergent evidence for NUDT17's subcellular localization, essential for understanding its physiological function.

How can NUDT17 antibodies support knockdown validation in functional studies?

NUDT17 antibodies serve as essential tools for validating knockdown efficiency in functional genomics studies:

• siRNA knockdown verification: After siRNA-mediated depletion of NUDT17 (similar to approaches used for other NUDIX hydrolases ), use Western blotting with NUDT17 antibodies to quantify protein reduction relative to control siRNA-treated cells.

• Quantitative verification: Implement densitometric analysis of Western blot bands, normalizing NUDT17 signal to housekeeping proteins (β-actin, GAPDH), aiming for >80% protein reduction to ensure phenotypic effects can be attributed to NUDT17 depletion.

• Temporal knockdown assessment: Use NUDT17 antibodies to track protein depletion kinetics at multiple timepoints post-transfection (24h, 48h, 72h, 96h) to identify optimal windows for phenotypic assays.

• Single-cell verification: Employ immunofluorescence with NUDT17 antibodies to assess knockdown efficiency at the single-cell level, important for transfection methods with variable efficiency.

• Rescue experiment design: In restoration experiments, use NUDT17 antibodies to confirm expression of siRNA-resistant NUDT17 constructs, ensuring they produce protein at physiological levels.

• Off-target effect assessment: For highly similar NUDIX family members (e.g., potential redundancy partners of NUDT17), use antibodies against these proteins to verify specificity of the knockdown.

• CRISPR-Cas9 knockout validation: When generating NUDT17 knockout cell lines, use antibodies to confirm complete protein elimination, distinguishing true knockouts from hypomorphic mutations.

• Inducible system calibration: For tetracycline-inducible or other conditional knockdown systems, use antibodies to calibrate inducer concentrations that achieve desired levels of NUDT17 depletion.

• Stable vs. transient knockdown comparison: Compare protein depletion efficiency between transient siRNA and stable shRNA approaches using quantitative Western blotting with NUDT17 antibodies.

• qPCR correlation: Compare mRNA reduction measured by qPCR with protein reduction detected by antibodies to identify potential post-transcriptional compensation mechanisms.

This methodical approach ensures that phenotypes observed after NUDT17 knockdown can be confidently attributed to specific depletion of this protein rather than off-target effects or incomplete knockdown.

What are the key considerations when using NUDT17 antibodies in cancer research?

When applying NUDT17 antibodies in cancer research contexts, consider these methodological factors:

• Expression heterogeneity: Unlike some NUDIX enzymes with consistent expression patterns across cancer types (e.g., NUDT1 being overexpressed in most cancers), NUDT17 may show variable expression . Use antibodies to systematically profile expression across diverse cancer types and compare with matched normal tissues.

• Tissue microarray analysis: Apply NUDT17 antibodies to cancer tissue microarrays containing multiple tumor types and stages to identify potential correlations with clinical parameters and outcomes.

• Cell line panel screening: Screen diverse cancer cell line panels (e.g., NCI-60) with NUDT17 antibodies to identify cancer types with particularly high or low expression for functional studies.

• Co-expression analysis: Investigate whether NUDT17 clusters with other NUDIX enzymes that have established roles in cancer, such as NUDT1, NUDT5, NUDT8, NUDT14, and NUDT22, which form a distinct cluster in cancer versus normal tissue comparisons .

• Survival correlation: Correlate NUDT17 protein levels (measured by antibody-based methods) with patient survival data to assess prognostic significance.

• Therapy response prediction: Determine whether NUDT17 expression levels correlate with response to specific therapies, particularly those inducing DNA damage or oxidative stress.

• Post-translational modification analysis: Use specific antibodies to investigate whether NUDT17 undergoes cancer-specific post-translational modifications that might alter its function.

• Synthetic lethality screening: Use antibodies to confirm NUDT17 levels when assessing potential synthetic lethal interactions with known cancer-associated genes, similar to approaches used with other NUDIX hydrolases .

• Subcellular localization changes: Determine whether NUDT17's subcellular distribution differs between normal and cancer cells, which might indicate altered function.

• Circulating tumor cell detection: Evaluate whether NUDT17 antibodies can be used to detect circulating tumor cells as part of liquid biopsy approaches.

This multifaceted approach leverages NUDT17 antibodies to comprehensively characterize this protein's role in cancer biology, potentially revealing new diagnostic or therapeutic opportunities.