NUDT9 Antibody

What is NUDT9 and what is its primary biological function?

NUDT9 (nudix nucleoside diphosphate linked moiety X-type motif 9) is a specific ADP-ribose (ADPR) pyrophosphatase that cleaves ADPR into AMP and ribose-5-phosphate. It belongs to the Nudix hydrolase superfamily, which catalyzes the hydrolysis of nucleoside diphosphates linked to other moieties . The protein has a calculated and observed molecular weight of 39 kDa .

NUDT9 contains two main subdomains:

• An N-terminal subdomain (cap) consisting of β-hairpins and α-helices

• A C-terminal subdomain (core) harboring the catalytic site with the highest homology to other Nudix hydrolases

While the isolated core subdomain retains activity and specificity for ADPR, the cap subdomain is also involved in substrate positioning . NUDT9 requires divalent metal ions and an intact Nudix motif for enzymatic activity .

What applications are NUDT9 antibodies typically used for?

NUDT9 antibodies are utilized across multiple research applications:

Different manufacturers recommend titrating the antibody in each testing system to obtain optimal results, as the dilution may be sample-dependent .

What is the species reactivity profile of available NUDT9 antibodies?

Available NUDT9 antibodies show variable reactivity across species:

When selecting an antibody for cross-species applications, researchers should consider sequence homology. For example, the immunogen used for NBP1-82715 displays 88% sequence identity with mouse NUDT9 and 89% with rat NUDT9 , suggesting potential cross-reactivity despite not being explicitly tested.

How should I optimize Western blot protocols for NUDT9 detection?

For optimal Western blot detection of NUDT9:

• Sample preparation:

  • Use fresh tissues or cell lysates from validated sources (e.g., mouse brain tissue, Raji cells, Jurkat cells)

  • Include appropriate positive controls (such as NUDT9 overexpression lysates)

• Protein loading and transfer:

  • Load 20-30 μg of total protein per lane

  • Use PVDF or nitrocellulose membranes with 0.45 μm pore size

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk in TBST

  • Use antibody dilutions in the recommended range (typically 1:500-1:3000)

  • Incubate primary antibody overnight at 4°C for optimal signal-to-noise ratio

• Detection and visualization:

  • Use appropriate HRP-conjugated secondary antibodies (e.g., Goat Anti-Rabbit IgG)

  • Expected band size: 39 kDa (both calculated and observed)

  • Typically requires 30 seconds exposure time with standard ECL systems

• Troubleshooting:

  • If detecting weak signals, increase antibody concentration or extend incubation time

  • For non-specific bands, increase blocking time or concentration of blocking agent

What are the recommended antigen retrieval methods for NUDT9 immunohistochemistry?

For successful IHC detection of NUDT9:

• Preferred antigen retrieval methods:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0

• Tissue preparation:

  • Fix tissues in 10% neutral buffered formalin

  • Embed in paraffin and section at 4-6 μm thickness

• Staining protocol:

  • Use antibody dilutions between 1:20-1:200 for IHC applications

  • Incubate with HRP-conjugated secondary antibody (typically 1:500 dilution)

  • Counterstain with hematoxylin for nuclear visualization

• Localization patterns:

  • Human kidney tissue: Cytoplasmic staining

  • Human heart and liver tissue: Positive detection reported

  • Rat kidney tissue: Cytoplasmic staining

For IHC-P applications using mouse-derived antibodies on mouse tissues, consider using Mouse-On-Mouse blocking reagents to reduce background signal .

How can I validate the specificity of a NUDT9 antibody?

A comprehensive validation strategy for NUDT9 antibodies should include:

• Positive and negative controls:

  • Positive tissue controls: Mouse brain, human kidney, human heart tissues

  • Positive cell line controls: Jurkat cells, HUVEC cells, Raji cells

  • Negative controls: PBS instead of primary antibody with identical secondary antibody treatment

  • Overexpression systems: HEK293T cells transfected with NUDT9 expression constructs

• Multiple detection methods:

  • Compare Western blot, IHC, and ICC/IF results for consistent detection patterns

  • Verify molecular weight matches the expected 39 kDa size

• Knockdown/knockout validation:

  • Use siRNA or CRISPR systems to reduce or eliminate NUDT9 expression

  • Confirm signal reduction proportional to protein reduction

• Peptide competition:

  • Pre-incubate antibody with immunizing peptide or recombinant NUDT9 protein

  • Observe elimination or significant reduction of specific signal

• Cross-reactivity assessment:

  • Test against closely related Nudix family members (especially NUDT10)

  • Verify specificity using orthogonal antibodies targeting different epitopes

How does NUDT9's role in ADP-ribose hydrolysis relate to TRPM2 channel function?

NUDT9 has significant implications for TRPM2 channel research:

• Structural and functional relationship:

  • TRPM2 contains a C-terminal NUDT9 homology (NUDT9-H) domain

  • This domain enables ADPR gating of the TRPM2 cation channel

  • NUDT9-H domain in TRPM2 shows reduced ADPRase activity compared to native NUDT9

• Key experimental findings:

  • TRPM2 splice variant lacking 34 amino acids in the NUDT9-H domain is insensitive to ADPR

  • Mutation I1405E/L1406F in the Nudix box of TRPM2 abolishes ADPR gating even at concentrations up to 10 mM

  • Deletion or substitution of Asn-1326 in TRPM2 abrogates ADPR gating

• Mechanistic insights:

  • Enhancing enzymatic activity of the Nudix box abolishes ADPR gating of TRPM2

  • This suggests prolonged binding rather than degradation is required for channel function

  • In invertebrate TRPM2 channels, ADPR hydrolysis is not coupled to pore gating

• Methodological approaches:

  • Use non-hydrolyzable ADPR analogs like AMPCPR to distinguish between binding and hydrolysis effects

  • Study TRPM2 channel currents with patch-clamp electrophysiology

  • Monitor ADPR hydrolysis with sensitive colorimetric assays that detect inorganic phosphate

These findings demonstrate that NUDT9 antibodies are valuable tools for investigating the relationship between ADPR metabolism and ion channel regulation.

What methodological considerations are important when studying NUDT9 subcellular localization?

When investigating NUDT9 subcellular localization:

• Sample preparation for immunofluorescence:

  • Fix cells with 4% paraformaldehyde

  • Permeabilize with 0.1% Triton X-100

  • Use appropriate blocking solutions to minimize non-specific binding

• Co-localization strategies:

  • NUDT9 shows mitochondrial localization in certain cell types

  • Use established mitochondrial markers like COX IV (cytochrome c oxidase subunit IV)

  • Perform dual staining with:

    • Anti-NUDT9 antibody (1:250 dilution)

    • Anti-COX IV antibody (1:1000 dilution)

• Detection systems:

  • Use fluorophore-conjugated secondary antibodies with minimal spectral overlap

  • For NUDT9: Alexa Fluor 488-conjugated anti-rabbit IgG

  • For mitochondrial markers: Alexa Fluor 594-conjugated anti-mouse IgG

• Controls and validation:

  • Include negative controls omitting primary antibodies

  • Use cross-controls (wrong secondary antibody) to verify specificity

  • Compare results from multiple fixation and permeabilization methods

• Advanced imaging techniques:

  • Super-resolution microscopy for precise co-localization assessment

  • Live-cell imaging with tagged NUDT9 constructs to confirm fixed-cell observations

  • Quantitative co-localization analysis using appropriate software and statistical methods

How can NUDT9 antibodies be utilized in studies examining oxidative stress responses?

NUDT9 antibodies can provide valuable insights into oxidative stress research through:

• Expression analysis during oxidative stress:

  • Monitor NUDT9 protein levels in response to various oxidative stressors

  • Compare expression patterns across different tissue and cell types

  • Correlate expression with markers of oxidative damage

• Relationship to TRPM2-mediated calcium signaling:

  • TRPM2 activation by ADPR is linked to oxidative stress responses

  • NUDT9 may regulate ADPR levels and thus influence calcium influx

  • Antibodies can help track this pathway in normal vs. stressed conditions

• Methodological approach:

  • Use Western blot to quantify NUDT9 expression changes (1:500-1:3000 dilution)

  • Perform IHC on tissues from oxidative stress models (1:20-1:200 dilution)

  • Combine with flow cytometry for cell-specific quantification (1:170 dilution)

• Experimental design considerations:

  • Include time-course analyses to capture dynamic changes

  • Compare mitochondrial and cytosolic fractions separately

  • Correlate with functional measures of ADPR metabolism

• Integration with other techniques:

  • Combine antibody-based detection with ADPR quantification assays

  • Use NUDT9 enzyme activity measurements alongside protein detection

  • Consider NUDT9 knockdown/overexpression to establish causality

How should I address non-specific binding issues with NUDT9 antibodies?

When encountering non-specific binding:

• Optimization strategies for Western blot:

  • Increase blocking time and concentration (try 5% BSA instead of milk)

  • Test different antibody dilutions within the recommended range (1:500-1:3000)

  • Include competing proteins (non-fat milk or BSA) in antibody dilution buffer

  • Increase washing duration and number of wash steps

  • Use freshly prepared buffers and reagents

• For immunohistochemistry applications:

  • Optimize antigen retrieval conditions (compare TE buffer pH 9.0 vs. citrate buffer pH 6.0)

  • Test different fixation protocols and times

  • Include additional blocking steps with normal serum from secondary antibody host species

  • Reduce primary antibody concentration and extend incubation time

  • For mouse tissues using mouse antibodies, employ Mouse-On-Mouse blocking reagents

• For immunofluorescence:

  • Include autofluorescence quenching steps

  • Use confocal microscopy to reduce out-of-focus signals

  • Prepare fresh fixatives and ensure complete permeabilization

  • Include peptide competition controls to identify specific signals

• Validation approaches:

  • Compare multiple NUDT9 antibodies targeting different epitopes

  • Include genetic knockdown controls

  • Test antibodies on tissues from NUDT9 knockout models if available

How do I interpret contradictory results from different NUDT9 antibody clones?

When facing contradictory results:

• Systematic analysis approach:

  • Document the exact experimental conditions used with each antibody

  • Verify antibody lot numbers and storage conditions

  • Compare the target epitopes of different antibodies used

• Technical considerations:

  • Different antibodies may recognize distinct protein isoforms or post-translational modifications

  • Monoclonal antibodies (like EPR15175) offer high specificity but may be sensitive to epitope masking

  • Polyclonal antibodies (like 15068-1-AP) recognize multiple epitopes but may show more cross-reactivity

• Experimental strategies to resolve contradictions:

  • Use genetic approaches (siRNA, CRISPR) to validate specificity

  • Perform immunoprecipitation followed by mass spectrometry

  • Compare results with orthogonal methods (e.g., mRNA quantification)

  • Test multiple positive and negative control samples

• Documentation and reporting:

  • Thoroughly document all antibody information (catalog number, lot, dilution)

  • Report complete experimental conditions when publishing results

  • Consider pre-registering experimental protocols to reduce bias

• Collaboration approach:

  • Consult with other laboratories using these antibodies

  • Contact manufacturers for technical support regarding specific applications

  • Consider multi-laboratory validation studies for critical findings

What are the best practices for quantifying NUDT9 expression in complex tissue samples?

For accurate quantification in complex tissues:

• Sample preparation considerations:

  • Ensure consistent tissue handling and processing

  • Consider laser capture microdissection for cell-type specific analysis

  • Include multiple biological replicates to account for variability

• Quantification methods for Western blot:

  • Use loading controls appropriate for the experimental context

  • Consider multiple loading controls (total protein stains plus housekeeping proteins)

  • Establish a linear dynamic range for detection

  • Employ digital image analysis with appropriate background subtraction

• Quantitative IHC approaches:

  • Use automated staining platforms when possible for consistency

  • Employ digital pathology systems for unbiased quantification

  • Include reference standards on each slide for normalization

  • Consider multiplex IHC to correlate NUDT9 with cell-type markers

• Flow cytometry for cellular heterogeneity:

  • Use intracellular staining protocols for NUDT9 (fixation with 2% paraformaldehyde)

  • Include appropriate isotype controls

  • Use multi-parameter analysis to identify specific cell populations

  • Report results as both percentage positive and median fluorescence intensity

• Standardization and normalization:

  • Establish inter-assay controls for longitudinal studies

  • Use relative quantification against stable reference samples

  • Consider absolute quantification using purified protein standards

  • Document and report all normalization steps and calculations

How can NUDT9 antibodies contribute to understanding ADPR metabolism in disease states?

NUDT9 antibodies offer multiple avenues for disease research:

• Metabolic disorders:

  • Quantitative proteomics using SWATH-MS has revealed sophisticated metabolic reprogramming in hepatocellular carcinoma tissues involving NUDT9

  • Antibodies can help validate and extend these findings in patient samples

• Neurodegenerative diseases:

  • NUDT9 is expressed in brain tissue and may influence neuronal calcium homeostasis via TRPM2

  • Immunohistochemistry with NUDT9 antibodies can map expression changes in disease models

• Inflammatory conditions:

  • ADPR metabolism affects oxidative stress responses and inflammation

  • Quantify NUDT9 expression in inflammatory cell populations using flow cytometry with intracellular staining protocols

• Cancer research applications:

  • Study NUDT9 expression across cancer types using tissue microarrays

  • Correlate expression with patient outcomes and treatment responses

  • Investigate potential associations with metabolic adaptations in tumors

• Methodological approaches:

  • Combine antibody-based detection with functional assays of ADPR metabolism

  • Develop multiplex detection systems for NUDT9 and related pathways

  • Integrate antibody-based findings with genetic and clinical data

What emerging technologies might enhance NUDT9 antibody applications in research?

Several technological advancements show promise for NUDT9 research:

• Advanced imaging techniques:

  • Super-resolution microscopy for precise subcellular localization

  • Expansion microscopy to visualize NUDT9 in relation to organelle structures

  • Live-cell imaging with NUDT9 antibody fragments or nanobodies

• Single-cell applications:

  • Antibody-based single-cell proteomics

  • Mass cytometry (CyTOF) for high-dimensional analysis of NUDT9 in relation to numerous other proteins

  • Spatial transcriptomics combined with antibody detection

• Proximity-based methodologies:

  • Proximity ligation assays to study NUDT9 protein interactions

  • APEX2 proximity labeling with NUDT9 antibodies for identifying transient interactions

  • Correlative light and electron microscopy for ultrastructural localization

• Functional antibody applications:

  • Development of conformation-specific antibodies to distinguish active vs. inactive NUDT9

  • Intrabodies for monitoring and manipulating NUDT9 in living cells

  • Antibody-drug conjugates for targeted manipulation of NUDT9-expressing cells

• High-throughput screening:

  • Automated image-based screening of NUDT9 expression

  • Microfluidic antibody-based assays for rapid analysis

  • Integrated multi-omics approaches correlating NUDT9 protein levels with transcriptomic and metabolomic data