NUDT5 Antibody, HRP conjugated

What is NUDT5 and what are its primary cellular functions?

NUDT5 (Nucleoside Diphosphate-linked Moiety X Motif 5) is a member of the NUDIX hydrolase family with dual enzymatic capabilities. It primarily functions as an ADP-sugar pyrophosphatase in the absence of diphosphate, hydrolyzing ADP-ribose to AMP and ribose-5'-phosphate. In the presence of diphosphate, it catalyzes the synthesis of ATP . NUDT5 demonstrates hydrolytic activity against various modified nucleoside diphosphates including ADP-ribose, ADP-mannose, ADP-glucose, 8-oxo-GDP, and 8-oxo-dGDP .

The protein's biological functions include:

• Maintaining NAD+ pools after DNA damage

• Preventing deleterious non-enzymatic ADP-ribosylation of proteins

• Nuclear ATP synthesis required for progestin-mediated chromatin remodeling, transcription, and tumor cell proliferation

• A scaffolding role in regulating purine de novo synthesis when adenosine is abundant

What sample types have been validated for NUDT5 antibody detection?

NUDT5 antibodies have been validated in multiple sample types across various applications as shown in the table below:

When planning experiments, it's advisable to start with these validated samples and optimize protocols for any additional sample types.

What are the recommended dilutions for different applications of NUDT5 antibody?

Optimal antibody dilutions vary by application technique. Based on validated protocols, the following dilutions are recommended:

Always perform a dilution series when using the antibody in a new experimental context to determine the optimal concentration that provides the best signal-to-noise ratio .

How can NUDT5 antibodies be used to investigate the dual roles of NUDT5 in ADP-ribose hydrolysis and ATP synthesis?

NUDT5 has a phosphorylation-dependent dual function: when dephosphorylated at Thr-45, it catalyzes ATP synthesis in the nucleus . To investigate these dual roles:

• Differential subcellular fractionation: Use HRP-conjugated NUDT5 antibodies to detect the protein in nuclear versus cytoplasmic fractions, correlating with its enzymatic role in each compartment.

• Phosphorylation-specific detection: Combine general NUDT5 antibodies with phospho-specific antibodies targeting Thr-45 to distinguish between the ATP-synthesizing versus hydrolytic forms.

• Activity-based colocalization: Design experiments that couple NUDT5 immunodetection with fluorescent ATP indicators to visualize ATP production in nuclear compartments.

• Chromatin association assays: Use chromatin immunoprecipitation followed by Western blot with HRP-conjugated NUDT5 antibodies to determine its association with transcriptionally active regions.

This approach provides insights into how NUDT5 contributes to nuclear ATP generation required for energy-consuming chromatin remodeling events .

What experimental approaches can distinguish between NUDT5's enzymatic activity and its non-enzymatic scaffolding functions?

Recent research suggests NUDT5 has a physical scaffolding role distinct from its enzymatic activity . To differentiate between these functions:

• Selective degradation versus inhibition: Compare phenotypes between:

  • NUDT5 enzymatic inhibition (using compounds like TH5427 or ibrutinib derivatives)

  • Complete protein degradation (using PROTAC approaches like dNUDT5)

  • Point mutations affecting catalytic activity but preserving structure

• Protein interaction studies: Use co-immunoprecipitation with HRP-conjugated NUDT5 antibody followed by mass spectrometry to identify interaction partners that may depend on the physical presence of NUDT5 rather than its catalytic activity.

• Structure-function assessment: Design rescue experiments with catalytically dead but structurally preserved NUDT5 mutants to determine which phenotypes require enzymatic activity versus protein presence.

• Temporal dynamics: Use live-cell imaging with fluorescently tagged NUDT5 combined with fixed-cell confirmation using HRP-conjugated antibodies to track protein localization during specific cellular processes.

These approaches can help determine if NUDT5's role in scenarios like adenosine-mediated toxicity stems from its enzymatic function or its protein scaffold properties .

How can one differentiate between NUDT5 and the closely related NUDT14 in experimental systems?

NUDT5 and NUDT14 share functional similarities as both hydrolyze ADPr, but they have structural differences that can be exploited experimentally :

• Selective antibody validation: Validate NUDT5 antibody specificity by:

  • Western blot comparison using recombinant NUDT5 and NUDT14 proteins

  • Testing in NUDT5 knockout/knockdown systems

  • Peptide competition assays with unique peptide sequences

• Structural binding differences: Crystallographic data reveals that while both enzymes have conserved binding motifs (Y17 and W34 in NUDT14; Y36 and W46 in NUDT5), they have distinct interaction patterns with inhibitors . Design experiments that exploit these differences.

• Selective inhibitor approach: Use selective inhibitors like TH5427 which shows specificity for NUDT5 over NUDT14 due to differences in R51 (present in NUDT5 but not conserved in NUDT14) .

• Isoform-specific knockdown: Combine selective knockdown with antibody detection to confirm specificity and understand the distinct roles of each protein.

A comprehensive approach incorporating multiple methods provides the most reliable differentiation between these closely related family members.

What are the optimal fixation and permeabilization methods for NUDT5 immunofluorescence in different cell types?

Fixation and permeabilization protocols significantly affect epitope accessibility and antibody binding. For NUDT5 detection:

• Paraformaldehyde fixation: 4% PFA for 15-20 minutes at room temperature preserves most epitopes while maintaining cellular architecture.

• Permeabilization options:

  • For nuclear NUDT5 detection: 0.2% Triton X-100 (5-10 minutes) provides good nuclear access

  • For cytoplasmic NUDT5: 0.1% saponin (10 minutes) offers more gentle permeabilization

• Cell type considerations:

  • For HeLa cells: Standard protocols have been validated

  • For breast cancer cells (MCF-7, T-47D): Additional antigen retrieval may be beneficial

  • For primary cells: Reduce fixation time to 10 minutes to preserve epitope accessibility

• Epitope masking prevention: If detecting post-translational modifications, consider methanol fixation (-20°C, 10 minutes) which can better preserve phosphorylation states.

Always include positive control cell lines like HeLa where NUDT5 detection has been validated .

How can non-specific binding be minimized when using HRP-conjugated NUDT5 antibodies?

Non-specific binding can compromise data quality. Implement these strategies to improve specificity:

• Blocking optimization:

  • For Western blots: 5% non-fat dry milk in TBST for 1 hour at room temperature

  • For immunohistochemistry: 10% normal serum (from the species of the secondary antibody) with 1% BSA

  • Consider adding 0.1-0.3% Triton X-100 to blocking solutions for better antibody penetration

• Antibody dilution optimization:

  • Start with the recommended range (1:2000-1:12000 for Western blot)

  • Perform a dilution series to determine optimal concentration

  • Incubate at 4°C overnight rather than at room temperature to reduce non-specific binding

• Washing protocols:

  • For HRP-conjugated antibodies, increase washing steps (5-6 washes of 5 minutes each)

  • Use 0.1% Tween-20 in TBS or PBS for effective removal of unbound antibody

• Validation controls:

  • Include NUDT5 knockout/knockdown samples as negative controls

  • Pre-absorb antibody with recombinant NUDT5 protein as a specificity control

• Signal enhancement alternatives:

  • If HRP background persists, consider tyramide signal amplification which allows for more dilute antibody use while maintaining signal strength

What are the recommended controls for validating NUDT5 antibody specificity in research applications?

Proper controls are essential for ensuring reliable and reproducible NUDT5 detection:

• Positive controls:

  • Recombinant NUDT5 protein

  • Cell lines with known NUDT5 expression (HeLa, MCF-7)

  • Tissues with validated expression (mouse liver)

• Negative controls:

  • NUDT5 knockout or knockdown samples

  • Secondary antibody-only controls

  • Isotype controls matched to the NUDT5 antibody

• Specificity controls:

  • Peptide competition assays using the immunizing peptide

  • Cross-reactivity assessment with recombinant NUDT14 protein

  • Western blot demonstrating single band at expected molecular weight (22 kDa)

• Application-specific controls:

  • For IHC: Include tissue sections known to be negative for NUDT5

  • For IF/ICC: Nuclear counterstaining to confirm expected subcellular localization

  • For pull-down assays: Input control and IgG control

• Orthogonal validation:

  • Confirm findings using multiple NUDT5 antibodies targeting different epitopes

  • Correlate protein detection with mRNA expression data

How can NUDT5 antibodies be utilized to study its role in cancer biology and potential as a therapeutic target?

NUDT5 has emerging significance in cancer biology, particularly in breast cancer where its overexpression has been suggested as a potential prognostic marker :

• Expression profiling:

  • Use HRP-conjugated NUDT5 antibodies for tissue microarray analysis across cancer types

  • Correlate expression with clinical outcomes and therapeutic responses

  • Perform Western blot quantification in paired normal/tumor samples

• Functional studies:

  • Combine NUDT5 detection with proliferation markers in immunofluorescence studies

  • Use proximity ligation assays to detect NUDT5 interaction with hormone receptors in breast cancer

  • Evaluate NUDT5 nuclear localization in response to hormone treatments

• Therapeutic targeting assessment:

  • Monitor NUDT5 levels and localization in response to inhibitor treatment

  • Use cell fractionation followed by Western blot to track changes in nuclear versus cytoplasmic NUDT5

  • Develop target engagement assays using NUDT5 antibodies to validate compound binding

• Resistance mechanism studies:

  • Investigate NUDT5 expression in resistant versus sensitive cell lines

  • Examine mutations that impair interaction with partners like PPAT that may contribute to antimetabolite resistance

  • Use immunoprecipitation with NUDT5 antibodies to identify altered interaction patterns in resistant cells

These approaches can help elucidate NUDT5's role in cancer progression and identify contexts where targeting NUDT5 might be therapeutically beneficial.

What methods can be used to study the interaction between NUDT5 inhibitors and the protein using antibody-based techniques?

Several antibody-based approaches can characterize NUDT5-inhibitor interactions:

• Cellular target engagement assays:

  • NanoBRET assays as described for inhibitors like ibrutinib and compound 9

  • Cellular thermal shift assays (CETSA) with subsequent Western blot using HRP-conjugated NUDT5 antibodies to detect thermal stabilization upon inhibitor binding

• Conformational change detection:

  • Epitope-specific antibodies that recognize regions near the binding site may show altered binding upon inhibitor engagement

  • FRET-based assays using fluorescently labeled antibodies to detect inhibitor-induced conformational changes

• Competition binding studies:

  • Competitive ELISA where plate-bound NUDT5 is detected with antibodies in the presence/absence of inhibitors

  • Flow cytometry-based competition assays in fixed/permeabilized cells

• Subcellular redistribution:

  • Immunofluorescence to track NUDT5 redistribution following inhibitor treatment

  • Subcellular fractionation with subsequent Western blot using HRP-conjugated antibodies

• Functional consequences:

  • Combine inhibitor treatment with detection of NUDT5 interaction partners

  • Assess changes in post-translational modifications of NUDT5 (e.g., phosphorylation at Thr-45) following inhibitor binding

These methods can help validate target engagement and understand the cellular consequences of NUDT5 inhibition, particularly for dual inhibitors of NUDT5/NUDT14 like compound 9 .

How might NUDT5 antibodies be employed to explore the newly discovered non-enzymatic roles of this protein?

Recent evidence suggests NUDT5 has non-enzymatic scaffolding functions that impact adenosine-mediated toxicity and purine de novo synthesis . Future research using NUDT5 antibodies could:

• Protein-protein interaction mapping:

  • Immunoprecipitation with NUDT5 antibodies followed by mass spectrometry

  • Proximity-dependent biotin labeling (BioID or APEX) combined with NUDT5 antibody validation

  • Co-immunoprecipitation studies specifically targeting interactions with purine synthesis pathway components

• Structure-function analysis:

  • Domain-specific antibodies to identify which regions of NUDT5 are critical for non-enzymatic functions

  • Immunofluorescence studies with wild-type and mutant NUDT5 constructs to track localization patterns

• Temporal dynamics:

  • Time-course studies following metabolic stress or adenosine challenge

  • ChIP-seq with NUDT5 antibodies to identify potential DNA-binding or chromatin association patterns

• Integrated multi-omics:

  • Correlate NUDT5 protein levels (by quantitative Western blot) with metabolomic profiles

  • Combine phosphoproteomics with NUDT5 immunoprecipitation to understand signaling pathways involving NUDT5

• Differential protein complex isolation:

  • Size-exclusion chromatography followed by Western blot with HRP-conjugated NUDT5 antibodies

  • Native PAGE analysis to preserve protein complexes, followed by antibody detection

These approaches could help distinguish between NUDT5's enzymatic and structural roles, potentially revealing new therapeutic opportunities.

What considerations should be made when designing experiments to study the phosphorylation state of NUDT5 and its impact on function?

NUDT5 phosphorylation, particularly at Thr-45, regulates its ability to synthesize ATP . When studying this regulation:

• Sample preparation considerations:

  • Use phosphatase inhibitors in all lysis buffers (10 mM sodium fluoride, 1 mM sodium orthovanadate, 10 mM β-glycerophosphate)

  • Perform rapid sample processing at cold temperatures to preserve phosphorylation state

  • Consider specialized phosphoprotein enrichment before antibody detection

• Antibody selection strategy:

  • Utilize phospho-specific antibodies targeting Thr-45 alongside total NUDT5 antibodies

  • Validate antibody specificity with lambda phosphatase-treated samples as controls

  • Consider developing custom phospho-specific antibodies if commercially available options lack specificity

• Detection method optimization:

  • For Western blots: Use PVDF membranes and enhanced chemiluminescence detection

  • For immunofluorescence: Methanol fixation often better preserves phospho-epitopes

  • For flow cytometry: Optimize permeabilization to maintain phosphorylation while allowing antibody access

• Functional correlation approaches:

  • Combine phospho-state detection with ATP synthesis assays

  • Use phosphomimetic and phospho-null mutants as controls

  • Perform kinase inhibitor treatments to manipulate phosphorylation state

• Quantification considerations:

  • Always normalize phospho-NUDT5 to total NUDT5 levels

  • Use ratiometric imaging approaches for immunofluorescence studies

  • Include calibration standards for accurate quantification

These methodological considerations ensure reliable detection of NUDT5 phosphorylation states and their correlation with functional outcomes.