NUDT11 Antibody

What is NUDT11 and why is it significant in research?

NUDT11, also known as diphosphoinositol polyphosphate phosphohydrolase 3-beta (DIPP-3-beta) or APS1, is an enzyme that primarily cleaves beta-phosphate from diphosphate groups in diphosphoinositol pentakisphosphate (PP-InsP5). It plays a significant role in signal transduction pathways and can catalyze the hydrolysis of dinucleoside oligophosphates, with Ap6A and Ap5A being preferred substrates . Recent research has implicated NUDT11 in prostate cancer pathogenesis, making it an important target for cancer research . The gene is located on the human X chromosome, which adds another dimension to its research significance in sex-linked expression studies .

What are the key applications for NUDT11 antibodies in research?

NUDT11 antibodies are versatile tools in molecular biology research with applications including:

• Western Blot (WB): Used at dilutions of 1:500-1:2000 or 1:1000-1:6000 depending on the specific antibody formulation

• Immunohistochemistry (IHC): Typically used at dilutions of 1:20-1:200 or 1:200-1:800

• Enzyme-Linked Immunosorbent Assay (ELISA): Used for quantitative detection of NUDT11 protein

• Immunocytochemistry/Immunofluorescence (ICC/IF): Employed for cellular localization studies

The choice of application depends on the specific research question, with Western blotting commonly used for protein expression analysis and IHC for tissue localization studies .

What are the different forms of NUDT11 antibodies available for research?

NUDT11 antibodies are available in several formats to accommodate different experimental needs:

• Polyclonal antibodies: Most commonly available, usually raised in rabbits, offering broad epitope recognition

• Tagged recombinant proteins: Includes options with Myc-DYKDDDDK Tag, His tag, or Strep Tag for various detection and purification methods

• Conjugated antibodies: Some are available with fluorescent tags (e.g., CoraFluor 1) for direct detection without secondary antibodies

• Species reactivity: Antibodies with confirmed reactivity to human, mouse, and/or rat NUDT11

The form selected should align with experimental goals, required sensitivity, and available detection systems in the laboratory .

How should NUDT11 antibody validation be performed for different applications?

Proper validation of NUDT11 antibodies is critical for ensuring experimental reliability:

• Western Blot validation: Confirm specificity by detecting a single band at the expected molecular weight (approximately 19 kDa for NUDT11) . Include positive controls such as brain tissue lysates from human, mouse, or rat specimens, which have demonstrated positive detection .

• IHC validation: Test antibody performance on known positive tissues such as mouse testis tissue or human ovary tumor tissue. Use appropriate antigen retrieval methods (suggested: TE buffer pH 9.0 or alternatively citrate buffer pH 6.0) .

• Cross-reactivity assessment: Since NUDT11 antibodies may recognize other NUDT family members (NUDT3, NUDT4, NUDT10) due to high homology, confirm specificity for your target of interest using knockout/knockdown controls or recombinant proteins .

• Titration experiments: Determine optimal dilutions by testing a range of concentrations in your specific experimental system rather than relying solely on manufacturer recommendations .

Complete validation should include positive and negative controls, assessment of background signal, and reproducibility testing across multiple experiments .

What are the optimal sample preparation techniques for NUDT11 detection?

Effective sample preparation is crucial for successful NUDT11 detection:

For Western Blot:

• Tissue lysate preparation: Homogenize samples in RIPA buffer supplemented with protease inhibitors

• Protein quantification: Use Bradford or BCA assay for standardization

• Loading control selection: GAPDH or β-actin are appropriate for most applications

• Denaturation conditions: Heat samples at 95°C for 5 minutes in SDS loading buffer

For IHC:

• Fixation: 10% neutral buffered formalin (24-48 hours)

• Processing: Standard paraffin embedding

• Sectioning: 3-5 μm thickness recommended

• Antigen retrieval: TE buffer pH 9.0 has shown superior results, with citrate buffer pH 6.0 as an alternative

• Blocking: 5% normal serum from the same species as the secondary antibody

For cell culture samples:

• Cells should be harvested at 70-80% confluence

• Wash with PBS to remove media components that may interfere with antibody binding

• Lysis buffers should be optimized based on subcellular localization of interest

What are the recommended dilutions and detection methods for NUDT11 antibodies?

Optimal dilution and detection parameters vary by application:

For Western Blot:

• Primary antibody dilution: 1:1000-1:6000, depending on antibody quality and sample type

• Secondary antibody: HRP-conjugated anti-rabbit IgG at 1:5000-1:10000

• Detection method: Enhanced chemiluminescence (ECL) systems work well for most applications

• Exposure time: Short exposures (30 seconds to 5 minutes) typically sufficient due to the 19 kDa size

For IHC:

• Primary antibody dilution: 1:200-1:800 for paraffin sections

• Secondary detection: Polymer-based detection systems provide higher sensitivity with lower background

• Chromogen: DAB (3,3'-diaminobenzidine) produces a brown precipitate that contrasts well with hematoxylin counterstain

• Counterstain: Hematoxylin (light application to avoid obscuring specific staining)

For ICC/IF:

• Primary antibody dilution: Begin with 1:100-1:500

• Secondary antibody: Fluorophore-conjugated anti-rabbit IgG at 1:200-1:1000

• Nuclear counterstain: DAPI at 1:1000

• Mounting medium: Anti-fade formulation to prevent photobleaching

Always titrate antibodies in each specific experimental system to determine optimal concentration .

How can specificity issues with NUDT11 antibodies be addressed?

NUDT11 antibodies may present specificity challenges due to homology with other NUDT family members:

• Cross-reactivity analysis: The antibody described in search result #1 can recognize multiple NUDT isoforms (NUDT3, NUDT4, NUDT10, and NUDT11) due to their high homology . To determine if this is problematic for your specific research:

  • Include knockout/knockdown controls where available

  • Pre-adsorb antibody with recombinant proteins of potential cross-reactive family members

  • Compare staining patterns with antibodies from different sources or those targeting different epitopes

  • Perform peptide competition assays to confirm binding specificity

• Sample-specific optimization: Different tissue types may require different antibody concentrations. Brain tissue has shown positive WB detection while testis and ovary tissues have demonstrated positive IHC results .

• Genetic validation: Consider using CRISPR-Cas9 edited cell lines with NUDT11 knockout as negative controls

• Western blot confirmation: For IHC or IF applications, validate findings with Western blot to confirm the detected protein is of the expected molecular weight (19 kDa) .

What factors affect NUDT11 antibody performance in experimental settings?

Multiple factors can influence NUDT11 antibody performance:

• Storage conditions: Antibodies should be stored at -20°C and are typically stable for one year after shipment. Repeated freeze-thaw cycles should be avoided by preparing aliquots .

• Buffer composition: Most NUDT11 antibodies are provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Changes in buffer composition can affect binding efficiency.

• Antigen retrieval methods: For IHC applications, TE buffer at pH 9.0 has shown better results than citrate buffer (pH 6.0) . Inappropriate antigen retrieval can lead to false negative results.

• Sample preparation: Overfixation of tissues or incorrect cell lysis procedures can mask epitopes.

• Antibody age: Degradation occurs over time, particularly with repeated freeze-thaw cycles.

• Detection systems: Secondary antibody selection should match the host species of the primary antibody (typically rabbit for available NUDT11 antibodies) .

• Background reduction: For high background, consider:

  • Increasing blocking concentration (5-10% normal serum)

  • Adding 0.1-0.3% Triton X-100 for membrane permeabilization

  • Including 0.05-0.1% Tween-20 in wash buffers

  • Using secondary antibodies pre-adsorbed against serum proteins from the sample species

How should researchers interpret and validate NUDT11 expression data?

Proper interpretation of NUDT11 expression data requires rigorous validation:

• Expression level baseline: NUDT11 has tissue-specific expression patterns. Brain tissue from human, mouse, and rat sources has shown positive Western blot detection . Establish appropriate positive and negative control tissues.

• Multiple detection methods: Confirm findings using orthogonal techniques (e.g., if using IHC, validate with Western blot or qPCR).

• Quantification methods: For Western blot, normalize NUDT11 signals to appropriate loading controls. For IHC, consider using digital image analysis with standardized scoring systems.

• Statistical analysis: Apply appropriate statistical tests based on sample size and distribution. For expression studies comparing different conditions, consider power analysis to determine adequate sample sizes.

• Reproducibility validation: Confirm key findings across multiple biological replicates and with different antibody lots if possible.

• Functional validation: Complement expression data with functional studies, such as the knockdown experiments described in search result #2, which revealed that suppression of NUDT11 inhibited proliferation/viability in specific cell lines by up to 63% .

How can NUDT11 antibodies be utilized in cancer research?

NUDT11 has emerged as a significant target in cancer research, particularly in prostate cancer studies:

• Expression correlation with cancer risk: Genetic and functional analyses have implicated NUDT11 in prostate cancer pathogenesis. Research has shown that risk variants rs5945619 are associated with increased expression of NUDT11 .

• Functional studies methodology: To investigate the role of NUDT11 in cancer:

  • Knockdown studies: siRNA or shRNA approaches have demonstrated that suppression of NUDT11 inhibits proliferation/viability by 63% in LNCaP cells (androgen-sensitive prostate cancer cell line)

  • Colony formation assays: NUDT11 knockdown decreased anchorage-independent colony formation by 86.3% in LNCaP cells and 56.9% in PC3 cells (androgen-independent prostate cancer line)

  • Cell line selection: Effects of NUDT11 modulation vary significantly between cell lines - showing strong effects in LNCaP but different patterns in PC3, suggesting context-dependent functions

• Potential as a biomarker: NUDT11 has been investigated as part of autoantibody-based biomarker panels for cancer detection, though more research is needed to establish clinical utility .

• Therapeutic target assessment: The significant decrease in cancer cell viability and colony formation following NUDT11 suppression suggests it may be a valuable therapeutic target .

What are the latest methodological advances in NUDT11 antibody applications?

Recent technological developments have expanded NUDT11 antibody applications:

• Fluorescent conjugation: Direct conjugation of fluorophores (e.g., CoraFluor 1) to NUDT11 antibodies enables direct detection without secondary antibodies, reducing background and enabling multiplex staining approaches .

• Recombinant technology: Highly pure recombinant NUDT11 proteins with various tags (His, Myc-DYKDDDDK, Strep) are now available, enabling more precise antibody validation and providing positive controls for assays .

• Host system diversity: NUDT11 proteins produced in different expression systems (HEK-293 cells, yeast, cell-free protein synthesis) offer advantages for different applications, with mammalian cell expression providing more native post-translational modifications .

• Active learning approaches: Emerging machine learning strategies for antibody-antigen binding prediction could potentially improve NUDT11 antibody design and selection, with recent studies showing that active learning algorithms can reduce the number of required experimental variants by up to 35% .

• Multi-omics integration: Combining NUDT11 antibody-based detection with genomic and transcriptomic data provides more comprehensive insights into its biological role and regulation.

How can NUDT11 antibodies be employed in studies of cell signaling pathways?

NUDT11's role in diphosphoinositol polyphosphate metabolism positions it as an important component in cellular signaling networks:

• Signal transduction analysis: NUDT11 cleaves beta-phosphate from diphosphate groups in PP-InsP5 (diphosphoinositol pentakisphosphate), suggesting involvement in inositol phosphate signaling pathways . Antibody-based approaches can help map these interactions through:

  • Co-immunoprecipitation (Co-IP) assays to identify binding partners

  • Proximity ligation assays (PLA) to confirm protein-protein interactions in situ

  • Phosphorylation-specific antibodies to examine regulation of NUDT11 activity

• Subcellular localization studies: Immunofluorescence with NUDT11 antibodies can reveal dynamic changes in localization in response to stimuli, providing insights into activation mechanisms.

• Signaling pathway perturbation: Combined use of NUDT11 antibodies with inhibitors of known signaling pathways can help position NUDT11 within signaling cascades:

  • Treat cells with pathway inhibitors and examine effects on NUDT11 expression/localization

  • Combine NUDT11 knockdown with pathway component overexpression to identify epistatic relationships

• Temporal dynamics: Time-course studies using NUDT11 antibodies following stimulation can reveal the kinetics of NUDT11 involvement in signaling responses.

• Cross-talk with other pathways: Given NUDT11's ability to hydrolyze dinucleoside oligophosphates like Ap5A and Ap6A, it may function at the intersection of multiple signaling pathways , which can be explored using multiplexed antibody-based detection methods.

NUDT11 Antibody Validation Data in Different Cell Lines

NUDT11 Antibody Technical Specifications

NUDT11 Family and Homology