NUDT15 Antibody

What is NUDT15 and why is it significant in thiopurine metabolism research?

NUDT15 (Nudix Hydrolase 15) is a nucleotide diphosphatase that plays a critical role in thiopurine drug metabolism. It dephosphorylates thiopurine active metabolites such as TGTP and TdGTP, preventing their incorporation into DNA and thus reducing cytotoxicity . NUDT15's significance in research stems from the discovery that genetic variants (particularly p.Arg139Cys, p.Arg139His, p.Val18Ile, and p.Val18_Val19insGlyVal) result in 74.4%–100% loss of nucleotide diphosphatase activity, leading to thiopurine intolerance in patients . This enzyme is especially relevant in Asian populations where NUDT15 variants are substantially overrepresented compared to TPMT variants, making NUDT15 their predominant genetic cause for thiopurine toxicity . Research using NUDT15 antibodies enables investigation of enzyme expression, localization, and interaction with metabolic pathways, providing insights into personalized medicine approaches for patients receiving thiopurine therapy.

How should researchers validate NUDT15 antibodies for Western blotting applications?

Rigorous antibody validation for NUDT15 Western blotting should follow this methodological approach:

• Positive and negative controls: Use cell lines with known NUDT15 expression levels. Human lymphoid cell lines are appropriate positive controls based on NUDT15 knockdown studies . Include NUDT15 knockdown cells (using shRNA) as negative controls.

• Recombinant protein controls: Run purified recombinant wildtype NUDT15 alongside variant proteins (p.Arg139Cys, p.Arg139His, etc.) to confirm antibody recognition of both wildtype and clinically relevant variants.

• Molecular weight verification: Confirm detection at the expected molecular weight (~18-20 kDa for human NUDT15).

• Optimization protocol:

  • Test multiple antibody dilutions (1:500 to 1:5000)

  • Evaluate various blocking solutions (5% milk vs. BSA)

  • Compare different incubation times and temperatures

  • Assess multiple detection methods (chemiluminescence vs. fluorescence)

• Cross-reactivity assessment: Test antibody against mouse NUDT15 if planning cross-species studies, noting that inhibitors like TH7755 show different binding affinities for human (IC₅₀ = 30 nM) versus mouse (IC₅₀ = 144 nM) NUDT15 .

What are the optimal immunohistochemistry (IHC) protocols for NUDT15 detection in tissue samples?

For successful NUDT15 immunohistochemistry, researchers should implement this methodological approach:

• Fixation optimization: Compare 10% neutral-buffered formalin (24h) versus 4% paraformaldehyde (12-24h) to determine which better preserves NUDT15 epitopes.

• Antigen retrieval methods: Test heat-induced epitope retrieval with citrate buffer (pH 6.0) versus Tris-EDTA buffer (pH 9.0), as NUDT15's structure contains disulfide bonds that may require different retrieval conditions.

• Blocking protocol: Use 5-10% normal serum from the species of secondary antibody origin with 0.1-0.3% Triton X-100 for permeabilization.

• Primary antibody optimization:

  • Test multiple dilutions (1:100-1:500)

  • Compare overnight incubation at 4°C versus 2-hour incubation at room temperature

• Signal amplification: Consider tyramide signal amplification for low-abundance NUDT15 detection.

• Positive control tissues: Include bone marrow, lymphoid tissues, and intestinal epithelium where thiopurine metabolism is clinically relevant.

• Counterstaining: Use DAPI for nuclear visualization to assess potential nuclear localization of NUDT15.

How can NUDT15 antibodies be used to study protein stability differences between wildtype and variant NUDT15?

Researchers can employ several antibody-dependent techniques to analyze NUDT15 variant stability:

• Pulse-chase immunoprecipitation:

  • Metabolically label cells expressing wildtype or variant NUDT15 with ³⁵S-methionine

  • Chase with non-radioactive medium for various time points (0-24h)

  • Immunoprecipitate NUDT15 using validated antibodies

  • Analyze by SDS-PAGE and autoradiography to determine protein half-life

• Cellular thermal shift assay (CETSA):

  • Heat intact cells expressing wildtype or variant NUDT15 at different temperatures (37-70°C)

  • Prepare cell lysates and analyze soluble NUDT15 by Western blotting

  • Generate melting curves to compare thermal stability

  • This approach revealed that NUDT15 variants (R139C, R139H, V18I, Val18_Val19insGlyVal) have lower thermal stability than wildtype NUDT15

• Differential stability with inhibitor binding:

  • Compare stabilization effects of inhibitors like TH7755 on wildtype versus variant NUDT15

  • Data indicates TH7755 increases thermal stability of all NUDT15 variants, with differential effects:

• Ubiquitination analysis:

  • Immunoprecipitate NUDT15 from cells treated with proteasome inhibitors

  • Probe Western blots with anti-ubiquitin antibodies

  • Compare ubiquitination patterns between wildtype and variant NUDT15

What methods can researchers use to investigate NUDT15 subcellular localization and how it differs between variants?

To study NUDT15 subcellular localization:

• Immunofluorescence microscopy:

  • Transfect cells with expression constructs for wildtype and variant NUDT15

  • Fix cells and immunostain using validated NUDT15 antibodies

  • Co-stain with organelle markers (DAPI for nucleus, MitoTracker for mitochondria, etc.)

  • Analyze colocalization using confocal microscopy

• Subcellular fractionation with Western blotting:

  • Prepare nuclear, cytoplasmic, and organelle fractions

  • Confirm fraction purity using compartment-specific markers

  • Detect NUDT15 in each fraction using Western blotting

  • Compare distribution patterns between wildtype and variant NUDT15

• Proximity ligation assay (PLA):

  • Use NUDT15 antibodies alongside antibodies for suspected interaction partners

  • PLA signals indicate proteins in close proximity (<40 nm)

  • Quantify and compare signals between wildtype and variant NUDT15

• Live-cell imaging:

  • Create fluorescent protein fusions with wildtype and variant NUDT15

  • Validate that fusion proteins retain enzymatic activity

  • Track localization in real-time using confocal microscopy

  • Correlate with antibody-based fixed-cell imaging to confirm findings

How can co-immunoprecipitation with NUDT15 antibodies identify novel protein interactions in thiopurine metabolism?

To identify NUDT15 interaction partners through co-immunoprecipitation:

• Antibody immobilization strategies:

  • Compare direct antibody immobilization versus protein A/G beads

  • For challenging interactions, consider crosslinking antibodies to beads

  • Test both N-terminal and C-terminal targeting antibodies as epitope accessibility may differ in complexes

• Lysis conditions optimization:

  • Test multiple lysis buffers with varying detergent strengths (NP-40, CHAPS, Triton X-100)

  • Compare salt concentrations (150-500 mM NaCl)

  • Add stabilizing agents (glycerol, protease inhibitors, phosphatase inhibitors)

• Interaction validation workflow:

  • Perform forward and reverse co-IP (using antibodies against both NUDT15 and suspected partners)

  • Include appropriate controls (IgG, lysate from knockdown cells)

  • Consider stimulation conditions (thiopurine treatment, oxidative stress)

• Mass spectrometry analysis:

  • Process immunoprecipitated complexes for LC-MS/MS

  • Filter against non-specific binders using database comparison

  • Validate top hits through targeted co-IP and functional assays

  • Look specifically for interactions with thiopurine metabolism enzymes

How can NUDT15 antibodies help elucidate the mechanism of thiopurine resistance in cancer therapy?

Researchers investigating thiopurine resistance mechanisms can employ NUDT15 antibodies in these advanced applications:

• Chromatin immunoprecipitation (ChIP) for DNA damage response:

  • Treat cells with thiopurines to induce DNA-TG incorporation

  • Perform ChIP using antibodies against DNA damage response proteins

  • Correlate findings with NUDT15 expression levels and variant status

  • This approach can reveal how NUDT15-dependent DNA-TG levels (which were 4.7 times higher in NUDT15 knockdown cells) influence DNA damage signaling

• Therapeutic resistance biomarker development:

  • Create tissue microarrays from thiopurine-responsive and resistant patient samples

  • Perform multiplex immunohistochemistry for NUDT15 and related pathway proteins

  • Develop scoring algorithms correlating expression patterns with clinical outcomes

  • Assess whether NUDT15 expression levels predict thiopurine response independently of genotype

• Functional interaction network mapping:

  • Combine antibody-based proteomics with CRISPR screening data

  • Identify synthetic lethal interactions with NUDT15 deficiency

  • Map NUDT15-dependent resistance pathways

  • Validate key nodes using combination drug treatments

• Pharmacodynamic biomarker development:

  • Develop assays to measure DNA-TG levels using antibodies against thioguanine-modified DNA

  • Correlate DNA-TG levels with NUDT15 genotype and thiopurine dosing

  • In clinical samples, DNA-TG levels strongly correlate with MP dosage (P=0.0024 in Singaporean cohort, P=2.2×10⁻⁴ in Japanese cohort)

  • The ratio of DNA-TG to MP dosage varies significantly by NUDT15 genotype, providing a potential biomarker for personalized dosing

What experimental approaches can resolve contradictory findings regarding NUDT15 enzymatic mechanisms?

To address conflicting data about NUDT15 enzymatic mechanisms:

• In vitro enzyme kinetics with purified components:

  • Express and purify wildtype and variant NUDT15 proteins

  • Measure enzymatic activities using multiple methodologies:

    • Malachite green phosphate detection

    • HPLC separation of reaction products

    • Mass spectrometry for product identification

  • Compare substrate specificities and kinetic parameters (Km, Vmax, kcat)

• Structure-function correlation using neutralizing antibodies:

  • Generate antibodies targeting different NUDT15 domains

  • Test their effects on enzymatic activity and substrate binding

  • Correlate findings with crystal structures of NUDT15-inhibitor complexes

  • Map critical residues for catalysis and substrate recognition

• Real-time intracellular metabolite tracking:

  • Develop metabolite sensors for thiopurine metabolites

  • Monitor metabolite flux in cells with different NUDT15 expression levels

  • Compare with direct measurements of cellular thioguanine nucleotides

  • NUDT15 knockdown in lymphoid cells shifts equilibrium toward TGTP, with 7.5-times higher TGTP to TGMP ratio and 6.2-times higher percentage of TGTP in total thioguanine nucleotides

• Mathematical modeling validation:

  • Develop computational models of thiopurine metabolism incorporating NUDT15

  • Test predictions experimentally using antibody-based NUDT15 quantification

  • Refine models based on experimental data from various cell types and genetic backgrounds

How can proximity-based proteomic approaches with NUDT15 antibodies identify transient interactions during thiopurine metabolism?

For studying transient NUDT15 interactions:

• BioID or TurboID enzyme fusion approach:

  • Create NUDT15-BioID fusion proteins (wildtype and variants)

  • Confirm enzymatic activity of fusion proteins

  • Purify biotinylated proteins after expression in cells

  • Identify proximity partners using mass spectrometry

  • Compare interaction landscapes between wildtype and variant NUDT15

• APEX2 proximity labeling:

  • Generate NUDT15-APEX2 fusion constructs

  • Add biotin-phenol substrate and H₂O₂ for rapid (1 min) proximity labeling

  • Purify biotinylated proteins and identify by mass spectrometry

  • This approach provides temporal resolution to capture dynamics

• Split-BioID for specific interaction contexts:

  • Fuse NUDT15 with one half of split-BioID

  • Fuse suspected interaction partners with complementary half

  • Reconstituted BioID activity indicates interaction

  • This method reduces background and increases specificity

• Crosslinking-immunoprecipitation protocol:

  • Treat cells with cell-permeable crosslinkers of varied lengths

  • Immunoprecipitate NUDT15 complexes under denaturing conditions

  • Identify crosslinked partners by mass spectrometry

  • Validate using reciprocal IPs with antibodies against identified partners

How can researchers accurately quantify NUDT15 expression levels in patient samples for personalized medicine applications?

For reliable NUDT15 quantification in clinical specimens:

• Droplet digital PCR (ddPCR) with antibody validation:

  • Develop ddPCR assays for absolute quantification of NUDT15 mRNA

  • Correlate mRNA levels with protein levels measured by antibody-based methods

  • Determine concordance between transcript and protein expression

  • Establish reference ranges for different tissue types and disease states

• Multiplexed immunoassay development:

  • Design sandwich immunoassays for NUDT15 protein quantification

  • Include internal calibration curves using recombinant standards

  • Validate assay performance metrics (LOD, LOQ, precision, accuracy)

  • Compare performance across different patient populations

• Single-cell analysis protocols:

  • Perform single-cell proteomics using index sorting with NUDT15 antibodies

  • Correlate with single-cell RNA-seq data

  • Identify cell type-specific expression patterns

  • Assess heterogeneity within patient samples

• Clinical validation studies:

  • Compare NUDT15 protein levels with genotype in patient cohorts

  • Correlate expression levels with clinical outcomes and thiopurine tolerance

  • Develop predictive algorithms combining genotype and protein expression data

  • In clinical studies, patients with low activity NUDT15 diplotypes showed significantly lower thiopurine tolerance across multiple cohorts (P = 0.021, 2.1 × 10⁻⁵, and 0.0054)

What are the methodological considerations for using NUDT15 antibodies in the development of therapeutic NUDT15 inhibitors?

For antibody applications in NUDT15 inhibitor development:

• Target engagement assays:

  • Develop cellular thermal shift assays (CETSA) using NUDT15 antibodies

  • Measure thermal stabilization of NUDT15 by inhibitor candidates

  • TH7755 increases the apparent aggregation temperature (Tagg) of cellular NUDT15 by 4°C, indicating strong target engagement

  • Use isothermal dose-response fingerprint CETSA to determine inhibitor potency

• Conformational antibody development:

  • Generate antibodies recognizing specific NUDT15 conformational states

  • Use these to detect inhibitor-induced conformational changes

  • Apply in high-throughput screening assays for novel inhibitors

• Pharmacodynamic biomarker assays:

  • Develop immunoassays to measure inhibition consequences

  • Quantify DNA-TG incorporation as a function of inhibitor concentration

  • TH7755 increases 6-thioguanine cytotoxicity starting at concentrations as low as 50 nM without demonstrating cytotoxicity when applied alone

• Resistance mechanism investigation:

  • Use NUDT15 antibodies to study adaptive responses to inhibition

  • Monitor changes in NUDT15 expression, localization, or post-translational modifications

  • Identify compensatory pathways activated upon NUDT15 inhibition