NUDT19 Antibody

What is NUDT19 and why is it significant for metabolic research?

NUDT19 (nudix nucleoside diphosphate linked moiety X-type motif 19), also known as RP2, is a 375 amino acid protein that functions as a coenzyme A diphosphatase, assisting in the hydrolysis of CoA esters. It belongs to the nudix hydrolase family and contains one nudix hydrolase domain . The protein has dual subcellular localization, being present in both peroxisomes and mitochondria, which suggests its involvement in multiple metabolic pathways .

NUDT19's significance stems from its role in CoA metabolism and its potential involvement in lipid homeostasis. Recent research has demonstrated that NUDT19 deletion affects lipid metabolism in kidney tissue, particularly under high-fat diet conditions . The protein utilizes magnesium and/or manganese as cofactors and is encoded by a gene mapping to human chromosome 19q13.11, a region with remarkably high gene density .

What should researchers know about selecting appropriate NUDT19 antibodies?

When selecting NUDT19 antibodies for research, consider the following methodological factors:

• Specificity validation: Verify that the antibody has been validated against NUDT19 using techniques such as western blotting with known positive cell lines (e.g., 293T, K562) and immunohistochemistry in relevant tissues (e.g., human liver cancer samples) .

• Clonality considerations: Polyclonal antibodies like those described in the search results may provide broader epitope recognition but potentially higher background. For quantitative studies, monoclonal antibodies might offer more consistent results between batches.

• Species reactivity: Currently available NUDT19 antibodies show reactivity to human NUDT19 . For studies in other species, cross-reactivity validation is essential.

• Application compatibility: Ensure the antibody is validated for your specific application. Current antibodies are verified for Western Blot (WB) at dilutions of 1:500-1:2000 and Immunohistochemistry (IHC) at dilutions of 1:25-1:100 .

• Molecular weight expectations: Be aware that the observed molecular weight may not always match the calculated MW (42 kDa) due to post-translational modifications or other factors affecting protein mobility in electrophoresis .

What are the optimal conditions for using NUDT19 antibodies in Western Blotting?

For optimal Western Blot results with NUDT19 antibodies, implement the following methodological approach:

• Sample preparation: Use verified positive controls such as 293T or K562 cell lysates, which have been confirmed to express detectable levels of NUDT19 .

• Loading concentration: Load 20-40 μg of total protein per lane for cell lysates or 60-80 μg for tissue homogenates to ensure adequate signal detection.

• Dilution optimization: Begin with the manufacturer's recommended range (1:500-1:2000) and optimize through titration experiments . For weaker signals, consider longer incubation times (overnight at 4°C) rather than higher antibody concentrations to minimize background.

• Blocking conditions: Use 5% non-fat dry milk in TBST for 1 hour at room temperature to minimize non-specific binding.

• Mobility considerations: Be prepared for potential discrepancies between calculated (42 kDa) and observed molecular weights. As noted in the product information, "the actual band is not consistent with the expectation" due to potential post-translational modifications or other factors affecting electrophoretic mobility .

• Detection sensitivity: For low abundance detection, consider using more sensitive detection systems such as enhanced chemiluminescence plus (ECL+) or fluorescent secondary antibodies.

How should researchers approach NUDT19 detection in immunohistochemistry studies?

For successful immunohistochemical detection of NUDT19:

• Tissue fixation: Use 10% neutral buffered formalin fixation for 24-48 hours, followed by paraffin embedding for optimal epitope preservation and morphology.

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) for 20 minutes, as this has been successful with verified samples such as human liver cancer tissue .

• Antibody dilution: Begin at the higher end of the manufacturer's recommended dilution range (1:25-1:100) and optimize based on signal-to-noise ratio .

• Detection system: Use a polymer detection system rather than avidin-biotin to minimize endogenous biotin interference, particularly in kidney and liver tissues where NUDT19 is predominantly expressed.

• Controls: Include both positive controls (human liver cancer) and negative controls (primary antibody omission and ideally tissues from Nudt19 knockout models if available) .

• Subcellular localization: When interpreting results, remember that NUDT19 localizes to both peroxisomes and mitochondria, so a punctate cytoplasmic staining pattern should be expected rather than diffuse cytoplasmic or nuclear staining .

How does NUDT19 deletion affect metabolic parameters in experimental models?

Research with Nudt19 knockout mice has revealed significant metabolic alterations, particularly in the context of high-fat diet (HFD) feeding:

• Lipid metabolism changes: Global metabolomics analysis identified 126 significantly altered metabolites in Nudt19-/- mice on HFD, with 55 increased and 71 decreased compounds . Pathway enrichment analysis revealed that processes related to lipid metabolism, particularly non-esterified fatty acids (NEFA), were significantly affected.

• Fatty acid profile alterations: Long-chain fatty acids exhibited a global decrease across multiple classes (essential, monounsaturated, polyunsaturated, and branched-chain fatty acids) in the kidneys of Nudt19-/- mice . This was correlated with reduced albumin reabsorption.

• Protein expression changes: Proteomics analysis identified 9 significantly altered proteins in the kidney cortex of Nudt19-/- mice, with 4 of 5 elevated proteins being associated with lipid metabolism . These included:

  • Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase (ECH1)

  • 3-ketoacyl-CoA thiolase B (THIKB)

  • Enoyl-CoA hydratase domain-containing protein 2 (ECHD2)

• CoA metabolism: Despite NUDT19's role in CoA metabolism, total CoA levels remained similar between Nudt19-/- and wild-type mice, suggesting compensatory mechanisms or localized effects that may not be detectable at the whole tissue level .

• Physiological outcomes: Nudt19-/- mice fed a HFD showed increased albuminuria, suggesting impacts on kidney function, and tended to have lower blood pressure values than wild-type males .

What experimental approaches can elucidate NUDT19's role in peroxisomal versus mitochondrial metabolism?

To investigate NUDT19's dual localization and differentiate its functions in peroxisomes versus mitochondria, researchers should consider these methodological approaches:

• Subcellular fractionation protocol:

  • Implement differential centrifugation to separate peroxisomal and mitochondrial fractions

  • Verify fraction purity using organelle-specific markers (e.g., catalase for peroxisomes, cytochrome c oxidase for mitochondria)

  • Quantify NUDT19 distribution using validated antibodies

  • Measure CoA hydrolase activity in each fraction to correlate with NUDT19 levels

• Organelle-targeted NUDT19 variants:

  • Generate constructs with modified targeting sequences that direct NUDT19 exclusively to either peroxisomes or mitochondria

  • Compare metabolic outcomes using metabolomics analysis similar to that employed in the knockout studies

  • Focus on acyl-CoA profiles in each compartment, particularly branched-chain acyl-CoAs which showed significant changes in the knockout model

• Proximity labeling approaches:

  • Employ APEX2 or BioID fusions with NUDT19 to identify proximal interacting partners in each organelle

  • This approach can reveal compartment-specific functions based on the protein interaction networks

• Metabolic flux analysis:

  • Implement stable isotope-labeled substrate tracing (e.g., 13C-labeled fatty acids)

  • Compare metabolic flux alterations in wild-type versus Nudt19-/- models to identify which metabolic pathways are primarily affected

  • Correlate with the global decrease in long-chain fatty acids observed in knockout models

What is the emerging evidence for NUDT19's role in disease progression and cancer?

Recent research has uncovered potentially significant roles for NUDT19 in disease contexts beyond its established metabolic functions:

• Kidney disease associations: Deletion of Nudt19 increases albuminuria in mice fed a high-fat diet, suggesting its potential involvement in kidney pathophysiology under metabolic stress conditions .

• Cancer metabolism connections: NUDT19 has been identified as part of a fatty acid metabolism-related gene signature with prognostic significance in prostate cancer, where its increased expression correlates with disease progression .

• Hepatocellular carcinoma (HCC) implications: While NUDT19 is barely detectable in normal mouse liver, its expression is markedly upregulated in HCC, where it activates the mTORC1/P70S6K signaling pathway, promoting proliferation and cell migration .

• Metabolic reprogramming effects: Functionally, knocking down Nudt19 in Hepa 1–6 cells enhances fatty acid oxidation and ATP production, suggesting that NUDT19 may be involved in the metabolic reprogramming characteristic of cancer cells .

• Therapeutic target potential: These findings collectively suggest that NUDT19 may represent a novel therapeutic target in certain cancers, particularly those with altered lipid metabolism.

When designing experiments to investigate NUDT19's role in disease models, researchers should consider:

• Correlating NUDT19 expression levels with disease progression markers

• Implementing both gain-of-function and loss-of-function approaches

• Examining downstream signaling effects, particularly in the mTORC1 pathway

• Investigating metabolic flux alterations using stable isotope tracing

How can researchers address inconsistent NUDT19 antibody results?

When encountering inconsistent results with NUDT19 antibodies, implement this systematic troubleshooting approach:

• Molecular weight variations: The observed molecular weight of NUDT19 may not match the calculated 42 kDa value. This discrepancy is acknowledged by manufacturers and may result from post-translational modifications or other factors affecting electrophoretic mobility . To address this:

  • Run longer SDS-PAGE gels for better resolution

  • Include positive control lysates from verified sources (293T, K562 cells)

  • Consider using multiple antibodies targeting different epitopes for confirmation

• Signal intensity optimization:

  • Antibody titration: Test dilutions within and beyond the recommended range (1:500-1:2000 for WB, 1:25-1:100 for IHC)

  • Increase protein loading: For weak signals, increase loading amounts rather than antibody concentration

  • Extended exposure times: For WB, use multiple exposure times to capture optimal signal-to-noise ratio

• Sample preparation considerations:

  • Use fresh samples when possible to avoid protein degradation

  • Include protease inhibitors in all buffers

  • For tissues with high protease activity (like kidney), minimize processing time at room temperature

• Storage and handling issues:

  • Store antibodies at -20°C as recommended, avoiding repeated freeze-thaw cycles

  • Prepare working aliquots to minimize freeze-thaw degradation

  • Note the 12-month validity period from manufacturers

What controls should be included when validating NUDT19 antibody specificity?

To rigorously validate NUDT19 antibody specificity, incorporate these essential controls:

• Positive controls: Include lysates from cell lines verified to express NUDT19, such as 293T and K562 cells for Western blotting, and human liver cancer samples for immunohistochemistry .

• Negative controls:

  • Primary antibody omission: Processes samples identically but omit primary antibody

  • Knockout/knockdown validation: Where available, tissues or cells with NUDT19 genetic ablation provide the most stringent specificity control

  • Pre-absorption control: Pre-incubate antibody with excess immunizing peptide to block specific binding

• Loading controls:

  • For Western blotting, include housekeeping proteins appropriate to the subcellular fraction being analyzed

  • For mitochondrial and peroxisomal proteins, use organelle-specific markers rather than global housekeeping genes

• Antibody validation across applications:

  • Confirm concordance between WB and IHC results when possible

  • For quantitative applications, demonstrate linearity of signal within the working range

• Cross-reactivity assessment:

  • Test specificity in tissues known to express low levels of NUDT19

  • In multi-species studies, confirm specificity for each species independently

What are promising areas for future investigation of NUDT19 function?

Based on current knowledge gaps and emerging evidence, these research directions represent promising avenues for future NUDT19 investigations:

• Peroxisomal-mitochondrial crosstalk: Investigate how NUDT19's dual localization might facilitate metabolic coordination between these organelles, particularly in tissues with high fatty acid oxidation requirements.

• Tissue-specific metabolic roles: NUDT19 is predominantly expressed in kidneys, but its upregulation in certain cancers suggests context-dependent functions . Comparative studies across tissues could elucidate these specialized roles.

• Regulation of NUDT19 expression: Little is known about how NUDT19 expression is regulated under different physiological and pathological conditions. Investigating transcriptional and post-transcriptional regulatory mechanisms could provide insights into its context-dependent functions.

• Therapeutic targeting potential: Given NUDT19's emerging roles in cancer metabolism and kidney function, developing specific inhibitors could have therapeutic applications. Structure-based drug design approaches could leverage the known nudix hydrolase domain .

• CoA homeostasis mechanisms: The finding that total CoA levels remain unchanged in Nudt19-/- mice despite its CoA diphosphatase activity suggests compensatory mechanisms that warrant further investigation .

How might metabolomics approaches enhance our understanding of NUDT19 function?

Advanced metabolomics strategies can provide deeper insights into NUDT19's metabolic roles:

• Targeted CoA metabolite profiling: Develop and implement sensitive methods for comprehensive profiling of CoA species in different subcellular compartments to better understand NUDT19's impact on CoA homeostasis.

• Stable isotope tracing: Implement 13C-labeled substrate tracing to track metabolic flux through pathways potentially affected by NUDT19, such as peroxisomal and mitochondrial β-oxidation.

• Spatial metabolomics: Apply emerging mass spectrometry imaging techniques to visualize metabolite distributions in tissues from wild-type and Nudt19-/- mice, potentially revealing localized metabolic alterations not detectable in whole-tissue analyses.

• Integration with multi-omics data: Correlate metabolomics findings with proteomics and transcriptomics data to build comprehensive network models of NUDT19-dependent metabolic regulation, extending the preliminary proteomics findings that identified alterations in lipid metabolism-related proteins .

• Temporal dynamics: Implement time-course metabolomics studies to understand how NUDT19 influences metabolic adaptation to changing nutrient conditions or stressors, particularly in the context of high-fat diet challenges where significant phenotypes have been observed .