NUDT19 Antibody
Description
Introduction to NUDT19 Protein and Its Antibodies
NUDT19 (nudix hydrolase 19) is a coenzyme A diphosphatase that mediates the hydrolysis of a wide range of CoA esters, including choloyl-CoA and branched-chain fatty-acyl-CoA esters. At low substrate concentrations, medium and long-chain fatty-acyl-CoA esters are the primary substrates for this enzyme . NUDT19 is a 375 amino acid protein that belongs to the nudix hydrolase family, containing one nudix hydrolase domain. This protein localizes to both mitochondria and peroxisomes and utilizes magnesium and/or manganese as cofactors for its enzymatic activity . Understanding NUDT19's function requires specific antibodies that can accurately detect this protein across various experimental platforms.
NUDT19 antibodies are immunoglobulins specifically designed to recognize and bind to the NUDT19 protein or its peptide fragments. These antibodies are essential tools in biochemical and cellular research, enabling scientists to investigate NUDT19's expression patterns, subcellular localization, and functional roles. Various commercial antibodies against NUDT19 are available, including both polyclonal and monoclonal variants with different host species and applications, making them versatile tools for scientific research in fields ranging from basic biochemistry to clinical studies .
Classifications Based on Production Method
NUDT19 antibodies are classified into two main types based on their production method: polyclonal and monoclonal antibodies. Polyclonal antibodies are derived from multiple B cell lineages in the host animal and recognize different epitopes on the NUDT19 protein . These antibodies offer high sensitivity due to their ability to bind multiple epitopes but may exhibit batch-to-batch variation. Monoclonal antibodies, conversely, are produced from a single B cell clone and recognize a single epitope on the NUDT19 protein . They provide consistent specificity and reproducibility across experiments but might have lower sensitivity compared to polyclonal antibodies.
Among the commercially available options, the rabbit recombinant monoclonal NUDT19 antibody from Abcam (ab180167, clone EPR13162-106) represents a modern approach combining the consistency of monoclonal antibodies with recombinant technology for enhanced reproducibility . Polyclonal variants, such as those from Thermo Fisher Scientific and Biofargo, offer alternative options for researchers with different experimental requirements .
Western Blotting Applications
Western blotting represents one of the most common applications for NUDT19 antibodies, enabling researchers to detect and quantify NUDT19 protein in cell or tissue lysates . In Western blot applications, NUDT19 antibodies typically detect a band at approximately 42 kDa, corresponding to the predicted molecular weight of the protein . Experimental validation has been reported using various cell lines, including K562 and 293T cells, as well as human ovary lysates . The recommended dilution range for Western blotting applications varies between 1:500 and 1:2000, depending on the specific antibody and experimental conditions .
Immunohistochemistry and Immunocytochemistry
NUDT19 antibodies are valuable tools for immunohistochemistry (IHC) and immunocytochemistry (ICC) applications, allowing researchers to visualize the spatial distribution of NUDT19 within tissues and cells . For IHC applications, human liver cancer tissue has been validated as a positive control, with recommended antibody dilutions ranging from 1:25 to 1:100 . These applications have revealed that NUDT19 primarily localizes to the cytoplasm, consistent with its function in metabolic processes involving CoA esters . Both polyclonal and monoclonal antibodies have demonstrated utility in these applications, though optimal conditions may vary between antibody types and experimental systems.
Additional Applications
Beyond the core applications described above, NUDT19 antibodies have demonstrated utility in several other experimental contexts:
These diverse applications highlight the versatility of NUDT19 antibodies as research tools across multiple experimental platforms, enabling comprehensive investigation of NUDT19 expression and function in various biological contexts.
Molecular Structure and Enzymatic Function
NUDT19 is characterized by its nudix hydrolase domain, which is essential for its enzymatic function . As a coenzyme A diphosphatase, NUDT19 mediates the hydrolysis of CoA esters by cleaving the diphosphate bond to form 3',5'-ADP and 4'-(acyl)phosphopantetheine . The enzyme shows substrate specificity for various CoA derivatives, with particularly high activity toward medium and long-chain fatty-acyl-CoA esters, as well as choloyl-CoA and branched-chain fatty-acyl-CoA esters . Interestingly, comparative studies have shown that while NUDT19 readily hydrolyzes short and medium chain acyl-CoAs, malonyl-CoA, succinyl-CoA, and free CoA, it exhibits poor activity against acetyl-CoA and synthetic monobromobimane (mBB)-CoA, which are excellent substrates for the related enzyme NUDT7 .
The enzymatic activity of NUDT19 depends on divalent metal ions, particularly magnesium and manganese, which serve as cofactors . Optimal activity for recombinant NUDT19 has been observed at 10 mM MgCl₂ when using free CoA as a substrate, although lower concentrations (4 mM) are sometimes necessary to prevent precipitation of certain acyl-CoA species such as lauroyl-CoA and stearoyl-CoA . Notably, NUDT19 shows specificity for CoA derivatives and has no detectable hydrolytic activity against other nucleotides like ATP, ADP, NAD⁺, NADH, NADP⁺, and NADPH .
Genetic Characteristics and Conservation
The NUDT19 gene is located on human chromosome 19q13.11, a region that spans approximately 63 million bases and houses about 1,400 genes . This chromosome is notable for having the greatest gene density among human chromosomes. The encoded NUDT19 protein consists of 375 amino acids and has a UniProt ID of A8MXV4 for the human protein . Alternative names for NUDT19 include RP2, Acyl-coenzyme A diphosphatase NUDT19, Nucleoside diphosphate-linked moiety X motif 19, nudix (nucleoside diphosphate linked moiety X)-type motif 19, and mitochondrial nucleoside diphosphate-linked moiety X motif 19 .
Tissue Distribution and Expression Patterns
Research using a polyclonal antibody against full-length mouse Nudt19 has revealed distinct tissue-specific expression patterns. Nudt19 protein shows the highest abundance in the kidneys, with lower but still detectable levels in skeletal muscle and brain . This expression pattern differs significantly from the related enzyme Nudt7, which is predominantly expressed in the liver . The kidney-specific expression of Nudt19 suggests specialized roles in renal metabolism and CoA homeostasis.
| Tissue | NUDT19 Expression Level | Reference |
|---|---|---|
| Kidney | High | |
| Skeletal Muscle | Moderate | |
| Brain | Low-Moderate | |
| Liver | Low/Undetectable |
This tissue-specific expression pattern aligns with functional studies suggesting that NUDT19 plays a critical role in regulating CoA levels specifically in the kidney . The differential expression of NUDT19 and the related enzyme NUDT7 across tissues indicates complementary roles in tissue-specific CoA metabolism.
In Vivo Studies and Knockout Models
Significant insights into NUDT19's physiological functions have emerged from studies using knockout mouse models. Research on Nudt19⁻/⁻ mice has demonstrated that deletion of the Nudt19 gene leads to elevated kidney CoA levels in mice fed ad libitum, providing direct evidence that NUDT19 contributes to the regulation of CoA in vivo . These findings confirm NUDT19's role as a kidney-specific regulator of CoA metabolism, distinguishing it from other CoA-degrading enzymes with different tissue distributions.
Biochemical Characterization
A notable difference between these enzymes lies in their ability to hydrolyze acetyl-CoA and synthetic monobromobimane (mBB)-CoA, which are excellent substrates for NUDT7 but poor substrates for NUDT19 . These biochemical distinctions, combined with the different tissue distributions of the two enzymes, suggest complementary roles in regulating CoA metabolism across different organs.
Implications for Metabolism and Disease
The regulation of CoA levels by NUDT19 has significant implications for cellular metabolism, particularly in the kidney. By controlling the availability of free CoA and various acyl-CoA species, NUDT19 potentially influences numerous metabolic pathways, including fatty acid oxidation, ketogenesis, and the citric acid cycle. The kidney-specific expression of NUDT19 suggests that this enzyme may have evolved to meet the unique metabolic demands of renal tissue .
While direct connections between NUDT19 dysfunction and specific diseases have not yet been thoroughly established, the enzyme's role in CoA metabolism suggests potential implications for metabolic disorders, particularly those affecting the kidney. Further research using NUDT19 antibodies will be essential for elucidating the enzyme's roles in health and disease, potentially revealing new therapeutic targets for metabolic and renal disorders.
Selection Criteria for Research Applications
When selecting a NUDT19 antibody for research purposes, several factors should be considered:
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Antibody Type: Polyclonal antibodies may offer higher sensitivity but potentially lower specificity compared to monoclonal antibodies. Recombinant monoclonal antibodies offer high consistency and reproducibility .
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Validated Applications: Different antibodies are validated for specific applications. Researchers should select antibodies that have been validated for their intended use, whether that's Western blotting, immunohistochemistry, ELISA, or other techniques .
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Species Reactivity: Ensure the antibody recognizes NUDT19 from the species under investigation. Most commercial antibodies target human NUDT19, but some may cross-react with mouse or rat orthologs .
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Epitope Information: Understanding which region of NUDT19 the antibody recognizes can be important, particularly for detecting specific isoforms or distinguishing between closely related proteins.
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Validation Data: Review the validation data provided by manufacturers, including Western blot images, immunocytochemistry results, and positive controls .
Practical Considerations
Practical aspects of antibody use should also be considered:
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Storage Conditions: NUDT19 antibodies typically require storage at -20°C in pH 7.4 PBS with 0.05% NaN3 and 40% Glycerol .
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Working Dilutions: Recommended dilutions vary by application and specific antibody. For Western blotting, dilutions typically range from 1:500 to 1:2000, while IHC applications may require 1:25 to 1:100 dilutions .
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Positive Controls: Knowledge of appropriate positive controls, such as human liver cancer tissue for IHC or K562 cells for Western blotting, can help validate experimental results .
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Cost and Quantity: Commercial antibodies are available in different quantities (e.g., 50 μl, 100 μl) at various price points (e.g., $149.00 for 50 μl from Biofargo) .
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- NUDT19 functions as an NADPH pyrophosphohydrolase, influencing cellular pyridine nucleotide levels and redox states. This modulation fine-tunes the photooxidative stress response. PMID: 27874073
- Research indicates that NUDT19 plays a crucial role in regulating the NADPH pool in plants, thereby impacting redox homeostasis. PMID: 26878367
Q&A
What is NUDT19 and what is its biological function?
NUDT19 (Nucleoside diphosphate-linked moiety X motif 19) is a fatty acyl-coenzyme A (CoA) diphosphatase that hydrolyzes fatty acyl-CoA to yield acyl-4'-phosphopantetheine and adenosine 3',5'-bisphosphate. It mediates the hydrolysis of a wide range of CoA esters, including choloyl-CoA and branched-chain fatty-acyl-CoA esters . At low substrate concentrations, medium and long-chain fatty-acyl-CoA esters are its primary substrates. NUDT19 shows highest activity with medium-chain acyl-CoA esters and demonstrates higher rates of activity with unsaturated acyl-CoA esters compared to saturated esters. It also exhibits decapping activity towards dpCoA-capped RNAs in vitro .
What are the common applications for NUDT19 antibodies in research?
NUDT19 antibodies are primarily used in the following applications:
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Western blotting (WB) for protein detection and quantification
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Immunohistochemistry on paraffin-embedded tissues (IHC-P)
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Immunocytochemistry/Immunofluorescence (ICC/IF)
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Flow cytometry (FC)
These applications enable researchers to study NUDT19 expression, localization, and function in various experimental contexts . When selecting an antibody, it's important to verify that it has been validated for your specific application and target species.
What controls should be included when using NUDT19 antibodies for Western blotting?
When using NUDT19 antibodies for Western blotting, include the following controls:
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Positive controls: Use tissue or cell lysates known to express NUDT19. Based on published research, kidney tissue (particularly from mice) shows high expression of NUDT19. Cell lines such as K562 and 293T have also been shown to express detectable levels of NUDT19 .
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Negative controls: Include samples from NUDT19 knockout models if available, or use tissue/cells with minimal NUDT19 expression. Comparative analysis with tissues from Nudt19−/− mice has been demonstrated to confirm antibody specificity .
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Loading controls: Standard housekeeping proteins such as β-actin, GAPDH, or tubulin should be probed to ensure equal loading across lanes.
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Antibody controls: Include a no-primary antibody control to assess non-specific binding of the secondary antibody.
Optimal dilution ratios generally range from 1:1000 to 1:10000 depending on the specific antibody and application, but always refer to the manufacturer's recommendations .
How can I validate NUDT19 antibody specificity in my experimental system?
To validate NUDT19 antibody specificity:
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Compare with genetic models: If possible, use tissues/cells from NUDT19 knockout or knockdown models. Western blotting analysis confirming the lack of Nudt19 protein expression in knockout mice has been successfully used to validate antibody specificity .
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Enzymatic assays: Confirm the correlation between antibody detection and enzymatic activity. For example, researchers have confirmed the lack of residual formation of 3′,5′-ADP in kidney homogenates from Nudt19−/− mice .
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Immunofluorescence co-localization: Since NUDT19 is peroxisomal, co-staining with established peroxisomal markers like PMP70 should show co-localization patterns .
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Pre-absorption controls: Pre-incubate the antibody with purified NUDT19 protein or the immunizing peptide before application to samples. This should abolish or significantly reduce specific staining.
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Cross-validation: Use multiple antibodies targeting different epitopes of NUDT19 and compare their detection patterns.
How can NUDT19 antibodies be used to investigate metabolic pathways in kidney disease models?
Recent research has shown that deletion of Nudt19 increases albuminuria in mice fed a high-fat diet, suggesting a role in kidney metabolism and disease . To investigate metabolic pathways using NUDT19 antibodies:
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Immunohistochemical profiling: Use NUDT19 antibodies to map expression changes in different kidney regions during disease progression. This can be combined with markers of peroxisomal function or kidney injury.
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Co-immunoprecipitation studies: Identify interaction partners of NUDT19 in kidney tissue using antibody-based pull-down assays followed by mass spectrometry. This can reveal novel metabolic network connections.
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Metabolic flux analysis: Combine NUDT19 protein quantification (via immunoblotting) with measurements of CoA species and fatty acid metabolism intermediates. In Nudt19−/− mice on high-fat diets, proteomics analysis revealed changes in proteins associated with lipid metabolism, including ECH1, THIKB, and ECHDC2 .
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Correlation with clinical markers: In translational studies, correlate NUDT19 expression levels with clinical parameters of kidney function and metabolic disease. Pathway-enrichment analysis has shown that processes related to lipid metabolism, particularly non-esterified fatty acids (NEFA), are significantly altered in kidneys of mice lacking NUDT19 .
What are the technical considerations for using NUDT19 antibodies in multi-parameter flow cytometry assays?
When incorporating NUDT19 antibodies in multi-parameter flow cytometry:
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Fixation and permeabilization: Since NUDT19 is an intracellular peroxisomal protein, proper fixation and permeabilization protocols are essential. Consider using commercially available peroxisome-specific permeabilization kits or optimize methanol-based protocols, which often work well for peroxisomal proteins.
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Antibody titration: Perform careful titration experiments to determine optimal antibody concentration, typically starting with dilutions between 1:10 and 1:50 for flow cytometry applications .
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Fluorophore selection: Choose fluorophores with minimal spectral overlap with other markers in your panel. If studying peroxisomal function, consider complementary markers like catalase or PEX proteins.
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Controls for intracellular staining: Include FMO (fluorescence minus one) controls and ensure isotype controls are properly matched. Consider using NUDT19 knockout cells as a definitive negative control if available.
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Compensation: Given the complexity of multiparameter flow cytometry, proper compensation is critical. Single-stained controls for each fluorophore should be included in each experiment.
What are common issues with NUDT19 antibody staining in immunohistochemistry and how can they be resolved?
Common issues and solutions:
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Weak or no signal:
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Increase antibody concentration or incubation time
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Optimize antigen retrieval methods (heat-induced or enzymatic)
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Verify tissue fixation protocols; overfixation can mask epitopes
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Use signal amplification systems such as avidin-biotin complex (ABC) or tyramide signal amplification (TSA)
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High background:
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Increase blocking time or concentration (typically 5% BSA or 10% normal serum)
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Optimize antibody dilution
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Include additional washing steps
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Use more specific secondary antibodies
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Pre-absorb antibody with non-specific proteins
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Non-specific staining:
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Verify antibody specificity using knockout controls
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Optimize primary antibody concentration
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Include additional blocking steps for endogenous peroxidase or biotin
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Use monoclonal antibodies instead of polyclonal if specificity is an issue
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Tissue-specific optimization:
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For kidney tissue, which has high NUDT19 expression, optimal dilutions may differ from those for other tissues
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Consider tissue-specific antigen retrieval methods (e.g., citrate buffer vs. EDTA)
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How do I resolve discrepancies between NUDT19 antibody results and mRNA expression data?
When facing discrepancies between protein and mRNA expression:
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Verify antibody specificity: Ensure the antibody is detecting only NUDT19 and not cross-reacting with related proteins. The Nudix hydrolase family contains multiple members with structural similarities that could lead to cross-reactivity.
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Consider post-transcriptional regulation: NUDT19 may be subject to post-transcriptional regulation, resulting in different levels of mRNA and protein. Investigate miRNA regulation or RNA stability factors.
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Evaluate protein turnover: Assess protein stability and degradation rates, which may explain differences between mRNA and protein levels. Consider pulse-chase experiments with protein synthesis inhibitors.
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Check for protein modifications: Post-translational modifications may affect antibody recognition. Try antibodies targeting different epitopes of NUDT19.
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Temporal considerations: mRNA and protein expression may peak at different times. Perform time-course experiments to capture these dynamics.
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Quantification methods: Evaluate the quantification methods used for both mRNA and protein. RT-qPCR might have different sensitivity compared to Western blotting. Consider absolute quantification methods for both.
How can NUDT19 antibodies be used to investigate the relationship between peroxisomal metabolism and kidney disease?
The 2025 study on Nudt19 deletion increasing albuminuria in mice fed a high-fat diet opens several research avenues :
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Histopathological mapping: Use NUDT19 antibodies alongside kidney injury markers to map the spatial distribution of peroxisomal changes in different kidney regions during disease progression.
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Cell-type specific analysis: Combine NUDT19 immunostaining with markers for specific kidney cell types (podocytes, tubular cells, etc.) to determine cell-specific alterations in peroxisomal metabolism.
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Mechanistic studies: Use NUDT19 antibodies to monitor changes in protein expression and localization in response to metabolic challenges. This can be correlated with the altered pathways identified in Nudt19−/− mice, particularly those related to lipid metabolism and fatty acid processing.
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Translational research: Develop immunohistochemical panels that include NUDT19 alongside other metabolic markers to assess peroxisomal function in human kidney biopsy specimens.
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Therapeutic response monitoring: Use NUDT19 antibodies to monitor peroxisomal responses to therapeutic interventions targeting metabolic dysfunction in kidney disease.
What methodological approaches can be used to study the interaction between NUDT19 and other enzymes involved in CoA metabolism?
To investigate NUDT19 interactions with other metabolic enzymes:
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Co-immunoprecipitation: Use NUDT19 antibodies to pull down protein complexes, followed by mass spectrometry or Western blotting for known CoA metabolic enzymes. This can identify direct protein-protein interactions.
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Proximity ligation assay (PLA): This technique can detect and visualize protein interactions in situ with high sensitivity. Combine NUDT19 antibodies with antibodies against potential interacting partners.
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FRET/BRET studies: For real-time interaction studies, develop fluorescent or bioluminescent protein fusions that can be monitored for resonance energy transfer when proteins interact.
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Immunofluorescence co-localization: High-resolution confocal microscopy with NUDT19 antibodies and antibodies against other peroxisomal enzymes can reveal spatial relationships within the peroxisome.
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Integrative multi-omics: Combine NUDT19 protein quantification with metabolomics of CoA species and lipidomics. This approach has revealed that long-chain fatty acids exhibit a global decrease across all classes in Nudt19−/− mice, correlating with reduced albumin reabsorption .
