NUDT5 Antibody, FITC conjugated

What is NUDT5 and what is its role in cellular metabolism?

NUDT5 (NUDIX hydrolase type 5), also known as NUDIX5, YSA1, YSA1H, hYSAH1, ADP-sugar pyrophosphatase, or HSPC115, is a 219 amino acid protein belonging to the nudix hydrolase family. This enzyme plays a critical role in cellular metabolism through its primary functions:

• Hydrolyzes ADP-ribose and ADP-mannose in the presence of magnesium

• Exhibits lower activity toward other nucleotide sugars like ADP-glucose and diadenosine diphosphate

• Eliminates potentially toxic nucleotide derivatives from cells

• Can either function as an ADP-sugar pyrophosphatase in the absence of diphosphate or catalyze ATP synthesis in the presence of diphosphate

NUDT5 functions as a homodimer and is predominantly expressed in the liver, indicating its importance in liver metabolism and broader physiological processes . Recent research has revealed that NUDT5 expression influences chromosome remodeling and is involved in cell adhesion, cancer stem cell maintenance, and epithelial-to-mesenchymal transition in breast cancer cells, suggesting its potential role in cancer progression .

What types of NUDT5 antibodies are available for research?

Researchers have multiple options for NUDT5 antibodies, varying in host species, clonality, and conjugation status:

When selecting an antibody, consider your experimental requirements including target species, application type, and whether you need a conjugated antibody for direct detection. The binding specificity can also vary, with some antibodies targeting specific amino acid regions (e.g., AA 34-166) of the NUDT5 protein .

What are the optimal applications for FITC-conjugated NUDT5 antibodies?

FITC-conjugated NUDT5 antibodies are particularly valuable for applications requiring direct fluorescence detection without secondary antibody incubation. Based on validation data, these antibodies excel in:

Immunofluorescence (IF)/Immunocytochemistry (ICC):

• Allows for direct visualization of NUDT5 cellular localization

• Optimal for HeLa cells, which show consistent positive detection with NUDT5 antibodies

• Recommended dilution ranges from 1:200-1:800 for optimal signal-to-noise ratio

Flow Cytometry:

• Enables quantitative analysis of NUDT5 expression in cell populations

• Particularly useful for comparing expression levels across different cell types or treatment conditions

Multiplex Immunofluorescence:

• FITC emission spectrum (peak ~520nm) allows combination with other fluorophores for co-localization studies

• Compatible with common nuclear counterstains like DAPI

When designing experiments using FITC-conjugated NUDT5 antibodies, consider phototoxicity and potential photobleaching of the FITC fluorophore during extended imaging sessions or repeated exposure to excitation light .

How should I determine the appropriate dilution for NUDT5 antibody experiments?

Determining optimal antibody dilution is crucial for balancing specific signal detection with background minimization. For NUDT5 antibodies, recommended dilutions vary by application:

To systematically determine optimal dilution:

• Perform a dilution series experiment with 3-4 concentrations within the recommended range

• Include positive controls (cells known to express NUDT5, such as HeLa, L02, MCF-7, MDA-MB-231, or T-47D cells)

• Include negative controls (secondary antibody only or isotype control)

• Evaluate signal intensity, specificity, and background

• Select the dilution that provides the strongest specific signal with minimal background

Remember that optimal dilution can be sample-dependent. For example, liver tissue samples (where NUDT5 is highly expressed) may require higher dilutions than tissues with lower expression .

What controls are essential when using NUDT5 antibodies in immunofluorescence studies?

Rigorous control experiments are critical for ensuring reliable and interpretable results with NUDT5 antibodies:

Essential Controls:

• Positive Control: Include cell lines with confirmed NUDT5 expression

  • HeLa cells: Consistently show positive detection in IF/ICC experiments

  • MCF-7, MDA-MB-231, and T-47D: Breast cancer cell lines suitable for NUDT5 studies

• Negative Controls:

  • Secondary antibody only: To assess non-specific binding of the secondary antibody

  • Isotype control: An antibody of the same isotype but irrelevant specificity

  • For FITC-conjugated antibodies: Unlabeled cells to establish autofluorescence baseline

• Specificity Controls:

  • Pre-absorption control: Pre-incubate antibody with excess recombinant NUDT5 protein

  • siRNA knockdown: Cells with NUDT5 expression reduced through RNA interference

  • CRISPR-Cas9 knockout: Cells with NUDT5 gene knockout for complete validation

• Technical Controls:

  • DAPI nuclear counterstain: To confirm cellular integrity and provide spatial reference

  • Cytoskeletal marker: To visualize cell boundaries and assess preservation of structure

These controls help distinguish between true NUDT5 signal and technical artifacts, particularly important when studying a protein like NUDT5 that may have varying expression levels across different cellular contexts .

How can I improve signal-to-noise ratio when using FITC-conjugated NUDT5 antibodies?

Optimizing signal-to-noise ratio is crucial for clear visualization of NUDT5 localization. Consider these methodological improvements:

Fixation and Permeabilization Optimization:

• For NUDT5 detection, 4% paraformaldehyde (10-15 minutes) followed by 0.1-0.2% Triton X-100 permeabilization (5-10 minutes) typically works well

• Methanol fixation may preserve certain epitopes better but can affect FITC fluorescence

Blocking Optimization:

• Use 5-10% normal serum from the same species as the secondary antibody

• Include 0.1-0.3% BSA and 0.1% Tween-20 in blocking solution

• Extend blocking time to 1-2 hours at room temperature

Antibody Incubation:

• Optimize dilution (typically 1:200-1:800 for NUDT5 IF applications)

• Incubate primary antibody overnight at 4°C to maximize specific binding

• For FITC-conjugated antibodies, protect from light during all steps

Washing:

• Increase number of washes (5-6 times, 5 minutes each)

• Use PBS with 0.1% Tween-20 for more effective removal of unbound antibody

Mounting and Imaging:

• Use anti-fade mounting medium specifically formulated for FITC

• Adjust exposure time to capture NUDT5 signal while minimizing background

• Consider deconvolution or confocal microscopy for improved resolution

Sample Preparation:

• For tissues requiring antigen retrieval, TE buffer pH 9.0 is recommended for NUDT5, with citrate buffer pH 6.0 as an alternative

Implementing these methodological refinements can significantly enhance the quality of NUDT5 immunofluorescence results across different experimental contexts .

What validation methods should I use to confirm NUDT5 antibody specificity?

Comprehensive validation of NUDT5 antibody specificity is essential for generating reliable research data. Implement these validation approaches:

Molecular Weight Verification:

• Compare observed molecular weight (35 kDa) with calculated weight (24 kDa) by Western blot

• Subtle differences may reflect post-translational modifications or protein complexes

Genetic Manipulation Approaches:

• siRNA knockdown: Confirm reduced signal following NUDT5 siRNA treatment

• CRISPR-Cas9 knockout: Generate NUDT5 knockout cells as definitive negative controls

• Overexpression: Confirm increased signal in cells transfected with NUDT5 expression vector

Epitope Mapping:

• For antibodies with defined epitopes (e.g., AA 34-166), verify recognition of recombinant fragments containing these regions

• Competitive binding assays with known NUDT5 substrates or inhibitors

Cross-Validation With Multiple Antibodies:

• Compare staining patterns using antibodies from different vendors or with different epitopes

• Both polyclonal (e.g., 27004-1-AP) and monoclonal (e.g., E-4) NUDT5 antibodies should show similar patterns

Functional Correlation:

• Correlate NUDT5 antibody staining with ADP-sugar pyrophosphatase activity

• Use the NanoBRET assay to confirm target engagement in live cells

These validation approaches collectively provide strong evidence for antibody specificity and support confident interpretation of experimental results involving NUDT5 detection .

How does NUDT5 function in normal versus cancer cells?

NUDT5 exhibits differential functions and expression patterns between normal and cancer cells, providing important research targets:

Normal Cells:

• Functions primarily as an ADP-sugar pyrophosphatase

• Hydrolyzes ADP-ribose and ADP-mannose in the presence of magnesium

• Eliminates potentially toxic nucleotide derivatives to maintain cellular homeostasis

• Predominantly expressed in liver tissue, functioning as a homodimer

• Plays roles in basic cellular metabolism and nucleotide pool maintenance

Cancer Cells:

• Shows altered expression levels, particularly in breast cancer

• Involved in chromosome remodeling processes

• Contributes to cell adhesion mechanisms

• Supports cancer stem cell maintenance

• Facilitates epithelial-to-mesenchymal transition in breast cancer cells

• High expression potentially correlates with poor prognosis in breast cancer patients

Experimental Approaches to Study These Differences:

• Comparative immunofluorescence using FITC-conjugated NUDT5 antibodies between normal and cancer cells

• Co-localization studies with chromatin markers to investigate remodeling functions

• Analysis of NUDT5 expression in paired normal/tumor tissue samples

• Functional studies using NUDT5 inhibitors like TH5 to assess cancer-specific vulnerabilities

Understanding these differences provides opportunities for developing targeted therapies and diagnostic approaches for cancers with NUDT5 dysregulation .

What cell lines are recommended for NUDT5 antibody validation?

Selecting appropriate cell lines is crucial for NUDT5 antibody validation. Based on positive detection data, these cell lines are recommended:

For tissue samples, mouse liver tissue has shown positive NUDT5 detection in IHC applications. When validating in this tissue, antigen retrieval with TE buffer pH 9.0 is recommended, with citrate buffer pH 6.0 as an alternative .

For advanced validation, consider using HEK293 cells transfected with NanoLuc-NUDT5 fusion constructs, which enable sensitive detection of target engagement in live-cell assays as demonstrated in recent studies of NUDT5 inhibitors .

How can NUDT5 antibodies help investigate epithelial-mesenchymal transition in cancer?

NUDT5's involvement in epithelial-mesenchymal transition (EMT) in breast cancer makes it a valuable target for studying cancer progression. FITC-conjugated NUDT5 antibodies enable several methodological approaches:

Co-localization Studies:

• Multiplex immunofluorescence combining NUDT5-FITC with antibodies against EMT markers:

  • E-cadherin (epithelial marker)

  • Vimentin (mesenchymal marker)

  • Snail/Slug/Twist (EMT transcription factors)

• Analyze spatial relationships between NUDT5 and these markers during EMT progression

Temporal Analysis:

• Track NUDT5 expression changes during induced EMT using models such as:

  • TGF-β treatment of epithelial cell lines

  • Hypoxia-induced EMT

  • Oncogene-driven EMT models

Functional Investigations:

• Combine NUDT5 immunofluorescence with invasion/migration assays

• Correlate NUDT5 expression/localization with invasive capacity

• Use NUDT5 inhibition to assess impact on EMT phenotypes

Clinical Sample Analysis:

• Apply NUDT5-FITC antibodies to patient-derived xenografts or tissue microarrays

• Analyze correlation between NUDT5 expression patterns and EMT status

• Compare primary tumors with metastatic lesions for NUDT5 expression changes

Since NUDT5 has been implicated in cancer stem cell maintenance, which often correlates with EMT, these approaches can provide valuable insights into the mechanisms underlying metastasis and therapeutic resistance in breast cancer .

What is known about NUDT5's interaction with BTK inhibitors and how can this be studied?

Recent research has revealed unexpected interactions between NUDT5 and BTK inhibitors, presenting new research opportunities:

Current Knowledge:

• Ibrutinib (1), an FDA-approved Bruton's tyrosine kinase (BTK) inhibitor used in cancer treatment, has been identified as a hit against NUDT5

• This discovery emerged from screening kinase inhibitors in an AMP-Glo assay monitoring NUDT5-mediated conversion of ADPr into AMP and ribose-5-phosphate

• Target engagement of ibrutinib with NUDT5 has been confirmed in-cell with an EC50 of 1.23 ± 0.10 μM

• Other BTK inhibitors did not show significant activity against NUDT5 (EC50 > 10 μM)

Methodological Approaches to Study This Interaction:

• NanoBRET Target Engagement Assay:

  • Utilize NanoLuc-NUDT5 fusion proteins expressed in HEK293 cells

  • Apply energy-transfer probes (ETFs) such as CBH-004 (compound 8)

  • Titrate BTK inhibitors to measure displacement of ETF and calculate EC50 values

  • Optimal assay conditions: 2.5 nM probe concentration with N-terminal NanoLuc-NUDT5 fusion

• Structural Analysis:

  • X-ray crystallography of NUDT5-ibrutinib complexes

  • Molecular docking to predict binding modes

  • Compare with known NUDT5-substrate structures (e.g., with ADP-ribose)

• Functional Impact Assessment:

  • Measure NUDT5 enzymatic activity in presence of BTK inhibitors

  • Investigate cellular consequences of dual BTK/NUDT5 targeting

  • Analyze potential synergies in cancer models

This unexpected cross-reactivity between BTK inhibitors and NUDT5 highlights the importance of comprehensive target profiling for kinase inhibitors and may reveal new therapeutic opportunities or mechanisms of action for these compounds .

How can I optimize immunofluorescence protocols specifically for NUDT5 detection?

Optimizing immunofluorescence protocols for NUDT5 detection requires attention to several key methodological aspects:

Fixation and Antigen Retrieval:

• For cell lines: 4% paraformaldehyde (15 minutes at room temperature) maintains both structural integrity and epitope accessibility

• For tissue sections: For NUDT5 IHC, TE buffer pH 9.0 is recommended for antigen retrieval, with citrate buffer pH 6.0 as an alternative

• For formalin-fixed paraffin-embedded samples: Heat-induced epitope retrieval is essential

Permeabilization:

• 0.1-0.2% Triton X-100 (10 minutes at room temperature) typically provides sufficient access to NUDT5 epitopes

• For nuclear NUDT5 detection, ensure complete nuclear membrane permeabilization

Blocking:

• 5% normal serum (from same species as secondary antibody) with 1% BSA in PBS

• 1-hour incubation at room temperature to minimize non-specific binding

Antibody Dilution and Incubation:

• For FITC-conjugated NUDT5 antibodies: 1:200-1:800 dilution range

• Primary incubation: Overnight at 4°C in humidified chamber (protected from light for FITC conjugates)

• For double-staining: Ensure antibodies are from different host species to prevent cross-reactivity

Counterstaining:

• DAPI (1 μg/ml, 5 minutes) for nuclear visualization

• Consider phalloidin-TRITC for cytoskeletal context if studying NUDT5 localization

Mounting:

• Anti-fade mounting medium specifically formulated for FITC preservation

• Allow mounting medium to cure completely before imaging (typically overnight at 4°C)

Imaging Parameters:

• Excitation: ~495 nm / Emission: ~520 nm for FITC detection

• Adjust exposure to avoid photobleaching while capturing sufficient signal

• Consider z-stack acquisition for complete cellular distribution analysis

What methods can be used to study NUDT5 enzyme activity alongside protein expression?

Comprehensive understanding of NUDT5 requires correlating protein expression with enzymatic activity. Consider these methodological approaches:

Parallel Analysis Techniques:

• AMP-Glo Assay:

  • Monitors NUDT5-mediated conversion of ADPr into AMP and ribose-5-phosphate

  • Quantitative luminescent readout correlating with enzyme activity

  • Can be used to screen inhibitors or assess activity in different conditions

• Immunofluorescence with Activity-Based Probes:

  • FITC-conjugated NUDT5 antibodies for protein localization

  • Activity-based probes that bind only to catalytically active NUDT5

  • Co-localization analysis reveals distribution of active versus inactive protein

• NanoBRET Target Engagement Assay:

  • Utilize N-terminal NanoLuc-NUDT5 fusion proteins

  • Apply energy-transfer probes like CBH-004 (compound 8)

  • Assess binding of substrates or inhibitors to NUDT5 in live cells

  • Optimal assay conditions: 2.5 nM probe concentration

• Combined Biochemical and Cellular Approaches:

  • Correlate in vitro enzyme kinetics with cellular localization

  • Analyze how mutations/modifications affect both activity and localization

  • Study how inhibitors affect both parameters simultaneously

• Visualization of Nucleotide Metabolism:

  • Use fluorescent analogs of ADP-ribose to track substrate utilization

  • Combine with NUDT5 immunostaining to correlate enzyme presence with activity

These integrated approaches provide deeper insights into NUDT5 biology than either expression or activity studies alone, particularly important when investigating NUDT5's dual functionality as either an ADP-sugar pyrophosphatase or ATP synthesizer depending on cellular conditions .

How can NUDT5 antibodies be used in multiplex imaging with other markers?

Multiplex imaging combining NUDT5 detection with other cellular markers provides contextual insights into NUDT5 function. Here's a methodological approach:

Compatible Marker Combinations:

Technical Considerations:

• Antibody Selection:

  • Choose primary antibodies from different host species to prevent cross-reactivity

  • For same-species antibodies, use directly conjugated antibodies or sequential immunostaining

• Spectral Separation:

  • FITC (peak emission ~520nm) pairs well with:

    • DAPI (~461nm)

    • Cy3/TRITC (~570nm)

    • Cy5/Alexa647 (~670nm)

  • Ensure sufficient spectral separation between fluorophores

• Optimization Strategy:

  • Titrate each antibody individually before combining

  • Validate specificity of each marker separately

  • Test for potential interference between antibodies

• Image Acquisition:

  • Use sequential scanning to minimize bleed-through

  • Optimize exposure settings for each channel

  • Include single-stained controls for spectral unmixing

• Analysis Approaches:

  • Co-localization analysis (Pearson's coefficient, Manders' overlap)

  • Population segmentation based on multiple markers

  • Spatial relationship mapping between NUDT5 and other proteins

This multiplex approach enables researchers to investigate NUDT5's functional relationships with cellular processes such as proliferation, differentiation, and cancer progression in a spatially resolved manner .

What emerging applications exist for NUDT5 research in cancer biology?

Recent discoveries about NUDT5 have opened several promising research directions in cancer biology:

Emerging Research Areas:

• NUDT5 in Hormone-Dependent Cancers:

  • NUDT5 has been implicated in breast cancer progression

  • High expression potentially correlates with poor prognosis

  • Research opportunities exist to investigate NUDT5's role in:

    • Estrogen receptor signaling

    • Hormone therapy resistance mechanisms

    • Cancer stem cell maintenance

• NUDT5 as a Therapeutic Target:

  • The discovery that BTK inhibitor ibrutinib interacts with NUDT5 (EC50 = 1.23 ± 0.10 μM) opens avenues for:

    • Repurposing existing drugs for NUDT5 inhibition

    • Structure-based design of specific NUDT5 inhibitors

    • Dual-targeting approaches for enhanced efficacy

• NUDT5 in Epithelial-Mesenchymal Transition:

  • NUDT5's involvement in EMT suggests research opportunities in:

    • Metastasis prevention strategies

    • Biomarker development for invasive disease

    • Combination approaches targeting EMT-related pathways

• NUDT5 in Chromosome Remodeling:

  • NUDT5's role in chromosome remodeling connects to:

    • Epigenetic regulation in cancer

    • DNA damage response pathways

    • Chromatin accessibility and gene expression control

• Functional Diversity of NUDT5:

  • NUDT5's dual role as either an ADP-sugar pyrophosphatase or ATP synthesizer suggests:

    • Context-dependent functions in different cancer types

    • Metabolic vulnerabilities that could be therapeutically exploited

    • Integration with cancer-specific metabolic adaptations

These emerging areas represent significant opportunities for researchers to advance understanding of cancer biology and develop novel therapeutic approaches targeting NUDT5-dependent processes .