UMPS Antibody, HRP conjugated

What is UMPS and why is it a target for antibody development?

UMPS (Uridine Monophosphate Synthetase), also known as OPRT and ODC, is a critical enzyme in the de novo pyrimidine biosynthesis pathway. It plays an essential role in nucleotide metabolism by catalyzing the conversion of orotic acid to uridine 5′ monophosphate (UMP), which represents a key step in pyrimidine biosynthesis. UMPS exists in four distinct isoforms with molecular weights of 53 kDa, 43 kDa, 33 kDa, and 23 kDa, though the predominant observed forms in experimental settings are typically 52 kDa and 45 kDa . Given its crucial role in cellular metabolism, UMPS is frequently targeted for antibody development to study pyrimidine synthesis pathways, investigate metabolic disorders, and explore its implications in various disease states, particularly cancer where nucleotide metabolism is often dysregulated .

What are the standard applications for UMPS antibodies in research?

UMPS antibodies have been validated for multiple experimental applications, making them versatile tools in molecular and cellular research. Western Blot (WB) analysis is the most common application, with recommended dilutions typically ranging from 1:500 to 1:2000 depending on the specific antibody preparation and experimental conditions . Immunohistochemistry (IHC) represents another important application, with dilutions generally between 1:20 and 1:200, allowing for detection of UMPS in fixed tissue samples such as human colon cancer tissue . Immunoprecipitation (IP) is also a validated application, requiring approximately 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate . Additionally, UMPS antibodies have been employed in ELISA assays for quantitative detection of the enzyme in complex biological samples . Researchers should consider that application-specific optimization is typically necessary, and published literature provides valuable reference points for experimental design.

How does HRP conjugation benefit antibody detection systems?

HRP (Horseradish Peroxidase) conjugation significantly enhances detection sensitivity in immunoassay systems through enzymatic signal amplification. Unlike direct labeling methods, the conjugation of HRP to antibodies enables enzymatic conversion of substrates like orthophenyldiamine, producing a detectable signal that can be substantially amplified compared to traditional detection methods . This enzymatic amplification can increase detection sensitivity by 15-fold or more, making it particularly valuable for detecting low-abundance targets like UMPS in complex biological samples . HRP conjugation also offers advantages in terms of stability, with properly prepared conjugates remaining functional for extended periods when stored appropriately. The versatility of HRP detection systems, which can be visualized using colorimetric, chemiluminescent, or fluorescent substrates, further contributes to their widespread adoption in research applications involving antibody-based detection strategies .

What are the reactivity profiles of commercially available UMPS antibodies?

Commercial UMPS antibodies demonstrate specific reactivity profiles that researchers should consider when selecting reagents for their experiments. Most UMPS antibodies show confirmed reactivity with human samples, making them suitable for clinical and biomedical research applications . Many antibodies also exhibit cross-reactivity with mouse UMPS, enabling comparative studies across human and murine models . When selecting a UMPS antibody, researchers should carefully review the validation data, which typically includes positive Western blot detection in cell lines such as HeLa, Jurkat, HEK-293T, and COLO 320 cells . For immunohistochemistry applications, positive reactivity has been documented in human colon cancer tissue, often requiring specific antigen retrieval conditions using TE buffer at pH 9.0 or citrate buffer at pH 6.0 . Cross-reactivity with other species should be experimentally validated if not explicitly stated in the product documentation, as sequence homology predictions may not always translate to functional cross-reactivity.

What are the optimal methods for conjugating HRP to UMPS antibodies?

The conjugation of HRP to UMPS antibodies requires careful consideration of chemical strategies to maintain antibody specificity while maximizing detection sensitivity. Heterobifunctional cross-linkers provide the most controlled approach for creating stable conjugates. The SATA-Sulfo-SMCC method represents a particularly effective strategy, wherein SATA (N-succinimidyl-S-acetylthioacetate) is first used to introduce protected sulfhydryl groups on the antibody, while Sulfo-SMCC is employed to activate HRP with maleimide groups . Following deprotection of the SATA-modified antibody to expose reactive thiols, the maleimide-activated HRP reacts specifically with these sulfhydryl groups to form stable thioether bonds, creating well-defined conjugates with preserved antibody activity . Alternative approaches include glutaraldehyde activation, which forms Schiff base linkages between primary amines on both the antibody and HRP . The optimal antibody-to-HRP ratio typically ranges from 1:2 to 1:4, though this must be empirically determined for each specific UMPS antibody preparation to balance detection sensitivity with potential steric hindrance that could compromise antigen binding .

How can researchers optimize signal amplification for low-abundance UMPS detection?

Detecting low-abundance UMPS protein requires strategic signal amplification approaches beyond standard HRP conjugation. Advanced poly-HRP systems represent a particularly effective strategy, wherein multiple HRP molecules are conjugated to each antibody molecule. One sophisticated method involves using a bromoacetylated peptide containing multiple lysine residues as a scaffold, which can be conjugated to SATA-modified antibodies or directly to reduced antibodies via 2-mercaptoethylamine (2-MEA) treatment . This approach introduces numerous reactive primary amines per antibody molecule, which can subsequently be coupled to maleimide-activated HRP, resulting in poly-HRP-antibody conjugates that deliver more than 15-fold signal amplification compared to conventional conjugates . For extremely challenging detection scenarios, researchers can employ tyramide signal amplification (TSA), wherein HRP catalyzes the deposition of biotinylated or fluorophore-labeled tyramide molecules at the site of antibody binding, creating a localized signal enhancement. Sequential multi-layer detection systems, employing primary anti-UMPS antibody followed by biotinylated secondary antibody and streptavidin-HRP, can also provide substantial amplification while maintaining specificity for UMPS detection in complex samples .

What are the critical considerations for optimizing UMPS antibody specificity in Western blot applications?

Achieving high specificity in Western blot applications using UMPS antibodies requires careful attention to several critical parameters. First, researchers should consider the multiple isoforms of UMPS (53 kDa, 43 kDa, 33 kDa, and 23 kDa) and anticipate potential detection of multiple bands, particularly at 52 kDa and 45 kDa as commonly observed in experimental settings . Blocking conditions significantly impact specificity, with 5% non-fat dry milk in TBST typically providing better background reduction than BSA-based blockers for UMPS detection. Antibody dilution optimization is essential, with most UMPS antibodies performing optimally between 1:500 and 1:2000, though this range should be empirically determined for each specific application and sample type . Researchers should also consider the epitope recognized by their selected UMPS antibody; for instance, the CAB5492 antibody targets amino acids 181-480 of human UMPS (NP_000364.1), which may influence detection of specific isoforms or fragments . Validation of specificity should include appropriate positive controls such as HeLa, Jurkat, HEK-293T, or COLO 320 cells, which have been confirmed to express detectable levels of UMPS . For challenging applications requiring absolute confirmation of specificity, parallel experiments using UMPS knockdown or knockout samples provide the most definitive validation.

How should researchers troubleshoot non-specific binding in UMPS immunohistochemistry?

Non-specific binding in UMPS immunohistochemistry can significantly compromise experimental interpretations and requires systematic troubleshooting approaches. Antigen retrieval conditions represent a critical starting point for optimization, with UMPS antibodies typically performing best using TE buffer at pH 9.0, though citrate buffer at pH 6.0 provides an alternative that may reduce background in some tissue types . Endogenous peroxidase activity, which can generate false positive signals with HRP-conjugated detection systems, should be quenched using 0.3% hydrogen peroxide in methanol for 30 minutes prior to primary antibody incubation. Non-specific protein interactions can be minimized by using species-matched normal serum (5-10%) from the same source as the secondary antibody, combined with BSA (1-3%) in blocking buffers. Titration of primary UMPS antibodies is essential, with optimal dilutions typically ranging from 1:20 to 1:200 for immunohistochemistry applications . For highly autofluorescent tissues, researchers should consider using HRP-conjugated detection with substrates like DAB rather than fluorescent methods, or employ Sudan Black B treatment to reduce tissue autofluorescence. When persistent non-specific binding occurs, peptide competition assays using the immunizing UMPS peptide sequence (such as amino acids 181-480 of human UMPS for the CAB5492 antibody) provide definitive evidence of antibody specificity .

How should researchers select appropriate control samples for UMPS antibody validation?

Rigorous validation of UMPS antibodies requires thoughtfully selected control samples that establish specificity and sensitivity parameters. Positive controls should include cell lines with confirmed UMPS expression, such as HeLa, Jurkat, HEK-293T, and COLO 320 cells, which have been documented to express detectable levels of UMPS protein . For tissue-based applications, human colon cancer tissue provides a reliable positive control for immunohistochemistry . Negative controls should incorporate both technical controls (primary antibody omission) and biological controls (tissues or cells with verified low or absent UMPS expression). For definitive specificity validation, researchers should consider employing UMPS knockdown or knockout models, which have been described in at least three publications referenced in the literature . When evaluating cross-reactivity across species, researchers should systematically test the antibody against human and mouse samples, as these represent the documented reactivity profile for most commercial UMPS antibodies . For antibodies targeting specific domains of UMPS, blocking peptide controls using the immunizing sequence (such as amino acids 181-480 of human UMPS for CAB5492) provide valuable confirmation of binding specificity . Researchers should document the performance of their selected antibody across this spectrum of controls before proceeding with experimental analyses.

What considerations are important when designing experiments to study UMPS in disease models?

Experimental design for studying UMPS in disease models requires careful consideration of multiple factors that impact interpretation and physiological relevance. When investigating UMPS in cancer models, researchers should acknowledge that pyrimidine metabolism is frequently dysregulated in transformed cells, potentially altering UMPS expression, localization, and function compared to normal tissues . Selecting appropriate control tissues is critical, ideally including both adjacent normal tissue and completely unrelated normal controls to establish baseline expression patterns. Temporal dynamics should be considered, particularly in models where UMPS activity might fluctuate with disease progression or treatment response. For metabolic disease studies, nutritional status significantly impacts nucleotide metabolism pathways, necessitating careful standardization of feeding/fasting conditions prior to sample collection. When employing genetic manipulation approaches (knockdown, knockout, or overexpression), researchers should verify the specificity of the intervention and assess potential compensatory mechanisms in pyrimidine synthesis pathways. Pharmacological studies targeting UMPS should include careful dose-response analyses and assessment of off-target effects. Translational studies should address potential species differences in UMPS regulation and function, particularly when extrapolating from murine models to human disease. Finally, researchers should consider how sample preparation might affect UMPS detection; for instance, rapid post-mortem changes can alter enzyme activities and protein degradation patterns, potentially confounding interpretations of UMPS expression and function in disease states .

How should researchers address variable signal intensity in UMPS Western blot analysis?

Variable signal intensity in UMPS Western blot analysis represents a common technical challenge that requires systematic troubleshooting. Sample preparation constitutes a primary consideration, with complete protease inhibition being essential to prevent degradation of UMPS, which can exist in multiple isoforms (53 kDa, 43 kDa, 33 kDa, and 23 kDa) . Extraction buffers should be optimized based on the cellular compartment being targeted, as UMPS distribution may vary across subcellular fractions. Protein loading consistency should be verified using total protein staining methods (such as Ponceau S or SYPRO Ruby) rather than relying solely on single housekeeping proteins, which may vary independently of UMPS. Transfer efficiency across the molecular weight range should be confirmed, particularly when comparing the multiple isoforms of UMPS, which span from 23 to 53 kDa. Antibody dilution represents another critical parameter, with most UMPS antibodies performing optimally between 1:500 and 1:2000; systematic titration experiments should be performed using positive control samples (such as HeLa or HEK-293T cell lysates) to establish the optimal concentration for each specific application . Incubation time and temperature significantly impact signal development, with overnight incubation at 4°C typically providing more consistent results than shorter incubations at room temperature. For HRP-conjugated detection systems, substrate incubation time should be standardized, and newer enhanced chemiluminescent substrates with extended signal stability should be considered for quantitative applications .

What approaches can resolve background issues in UMPS immunohistochemistry?

Resolving background issues in UMPS immunohistochemistry requires a methodical approach addressing multiple potential sources of non-specific signal. Antigen retrieval conditions should be carefully optimized, with UMPS antibodies typically performing best with TE buffer at pH 9.0, though citrate buffer at pH 6.0 provides an alternative that may reduce background in some tissue types . Endogenous peroxidase activity must be thoroughly quenched, particularly in tissues rich in peroxidases such as liver and kidney, using 0.3-3% hydrogen peroxide in methanol for 15-30 minutes prior to antibody incubation. For tissues with high endogenous biotin, which can cause background with avidin-biotin detection systems, biotin blocking kits or direct HRP-conjugated secondary antibodies should be employed. Non-specific protein binding can be minimized using carefully selected blocking reagents; for UMPS detection, 5-10% normal serum from the same species as the secondary antibody, combined with 1-3% BSA, typically provides effective blocking. Antibody dilution optimization is essential, with immunohistochemistry applications of UMPS antibodies typically requiring dilutions between 1:20 and 1:200 . Washing protocols should include detergent (0.05-0.1% Tween-20) in PBS or TBS buffers and sufficient duration (three washes of 5 minutes each) to remove unbound antibody effectively. For persistent background issues, researchers should consider polymer-based detection systems, which typically generate less background than traditional avidin-biotin methods, particularly in tissues with high endogenous biotin or when using rabbit polyclonal antibodies like those commonly employed for UMPS detection .

How can researchers validate the specificity of HRP-conjugated UMPS antibodies?

Validating the specificity of HRP-conjugated UMPS antibodies requires a multi-faceted approach combining molecular, biochemical, and immunological methods. Peptide competition assays represent a fundamental validation strategy, wherein pre-incubation of the antibody with excess immunizing peptide (such as the sequence corresponding to amino acids 181-480 of human UMPS for CAB5492) should abolish specific binding . Genetic validation provides the most definitive evidence of specificity, comparing detection in wild-type samples versus those with UMPS knockdown or knockout; at least three publications have employed this approach for UMPS antibody validation . Immunoprecipitation followed by mass spectrometry identification can confirm that the antibody captures the intended UMPS protein rather than cross-reactive species. Western blot analysis should demonstrate bands of appropriate molecular weight, primarily at 52 kDa and 45 kDa, though researchers should be aware of the potential to detect additional isoforms at 53 kDa, 43 kDa, 33 kDa, and 23 kDa depending on the specific antibody and sample type . Cross-reactivity testing against related proteins in the pyrimidine synthesis pathway can identify potential non-specific interactions. When evaluating HRP conjugation effects on specificity, parallel testing of the unconjugated primary antibody using secondary detection should yield identical patterns of reactivity, though potentially with different signal intensity. For quantitative applications, researchers should establish standard curves using recombinant UMPS protein to validate both specificity and the quantitative accuracy of the HRP-conjugated antibody system .

What strategies can overcome limited sensitivity in detecting low-abundance UMPS in complex samples?

Detecting low-abundance UMPS in complex samples requires specialized approaches to enhance sensitivity while maintaining specificity. Sample enrichment represents a primary strategy, with immunoprecipitation using unconjugated UMPS antibody being particularly effective; this approach has been validated for HEK-293 cells and can be adapted for other sample types . For tissue samples, laser capture microdissection can isolate specific regions with higher UMPS expression, increasing the relative abundance in the extracted sample. Signal amplification techniques significantly enhance detection limits, with poly-HRP systems being particularly effective; conjugating multiple HRP molecules to each antibody using bromoacetylated peptide scaffolds containing multiple lysine residues can increase sensitivity by more than 15-fold compared to conventional conjugates . Tyramide signal amplification (TSA) provides another powerful approach, wherein HRP catalyzes the deposition of additional reporter molecules at the site of antibody binding. For Western blot applications, enhanced chemiluminescent substrates with extended sensitivity should be employed, combined with longer exposure times on highly sensitive detection systems. When working with particularly challenging samples, researchers should consider sequential multi-layer detection systems, employing primary anti-UMPS antibody followed by biotinylated secondary antibody and streptavidin-poly-HRP complexes. Researchers should carefully balance sensitivity enhancement with the potential for increased background, validating each modification to ensure that the improved detection limit does not come at the cost of reduced specificity for UMPS .

How can UMPS antibodies be employed to investigate pyrimidine metabolism in cancer research?

UMPS antibodies provide valuable tools for investigating dysregulated pyrimidine metabolism in cancer, offering insights into both basic biology and potential therapeutic targeting. Immunohistochemical analysis using UMPS antibodies enables spatial evaluation of enzyme expression across different regions of tumors, revealing heterogeneity that may impact treatment response; human colon cancer tissue has been specifically validated for such applications . Correlation of UMPS expression with proliferation markers can establish connections between pyrimidine synthesis capacity and tumor growth dynamics. For mechanistic studies, co-immunoprecipitation using UMPS antibodies can identify novel protein-protein interactions that regulate enzyme function in cancer cells, with HEK-293 cells providing a validated system for developing such protocols . Combining UMPS protein detection with metabolomic analysis of pyrimidine intermediates can establish functional consequences of altered expression patterns. For therapeutic development, UMPS antibodies can monitor target engagement and pathway modulation following treatment with nucleotide metabolism inhibitors. Multi-parameter flow cytometry incorporating UMPS antibodies enables correlation of enzyme expression with cell cycle status and other cancer-relevant phenotypes at the single-cell level. When investigating resistance mechanisms, comparative analysis of UMPS expression and localization in sensitive versus resistant cell populations can identify adaptations in pyrimidine metabolism that contribute to treatment failure. Researchers should consider that UMPS dysregulation in cancer occurs within a complex network of metabolic alterations, necessitating integrated analysis with other components of nucleotide synthesis pathways .

What approaches enable quantitative analysis of UMPS enzyme levels across different experimental systems?

Quantitative analysis of UMPS enzyme levels requires carefully validated methodologies tailored to specific experimental systems. Enzyme-linked immunosorbent assays (ELISA) using UMPS antibodies provide the most precise quantification, though researchers must generate standard curves using recombinant UMPS protein of known concentration and validate the linear detection range for their specific sample types . Western blot densitometry offers semi-quantitative data suitable for comparative studies, with normalization to total protein loading being preferred over single housekeeping proteins, which may vary independently of UMPS; this approach has been validated for multiple cell lines including HeLa, Jurkat, HEK-293T, and COLO 320 cells . Mass spectrometry-based proteomics using isotope-labeled standards enables absolute quantification of UMPS protein, though this requires specialized equipment and expertise. For single-cell resolution, quantitative immunofluorescence microscopy combined with image analysis algorithms can measure UMPS levels while preserving spatial information. When comparing UMPS levels across different experimental systems, researchers should develop normalization strategies that account for matrix effects and ensure that samples fall within the validated linear range of the assay. For longitudinal studies, reference standards should be included in each experimental batch to control for inter-assay variation. Researchers should acknowledge that protein levels may not directly correlate with enzyme activity; therefore, complementary functional assays measuring the conversion of orotic acid to UMP provide important contextual information for interpreting quantitative protein data .

How can researchers integrate UMPS protein analysis with functional metabolic studies?

Integrating UMPS protein analysis with functional metabolic studies requires coordinated experimental approaches that connect enzyme levels with pathway activities. Targeted metabolomics measuring pyrimidine intermediates (particularly orotic acid and UMP) provides direct evidence of UMPS functional impact, with observed metabolite ratios reflecting enzyme activity in situ. Isotope tracing experiments using labeled precursors (such as 13C-labeled glucose or glutamine) can quantify flux through the UMPS-catalyzed step of pyrimidine synthesis, with the incorporation rate of labeled carbon into UMP directly reflecting enzyme function. Correlation of UMPS protein levels measured by Western blot or immunohistochemistry with metabolite concentrations across diverse samples can establish quantitative relationships between enzyme abundance and pathway activity. For mechanistic studies, comparative analysis following UMPS knockdown, knockout, or overexpression provides causal evidence linking protein levels to metabolic outcomes. Inhibitor studies using compounds that target steps upstream or downstream of UMPS can reveal regulatory relationships within the pathway. Technical considerations include the need for rapid sample processing to prevent post-collection metabolism, with direct quenching of enzymatic activities being essential for accurate metabolite quantification. Researchers should also consider that subcellular localization of UMPS, not just total protein levels, may impact functional activity; therefore, fractionation studies combined with Western blot analysis can provide important insights into the pool of enzyme actively engaged in pyrimidine synthesis .

What considerations are important when studying post-translational modifications of UMPS using antibody-based methods?

Studying post-translational modifications (PTMs) of UMPS using antibody-based methods requires specialized approaches that preserve modification status while enabling specific detection. Sample preparation represents a critical consideration, with rapid processing and inclusion of appropriate inhibitors (phosphatase inhibitors for phosphorylation studies, deubiquitinase inhibitors for ubiquitination analysis, etc.) being essential to prevent artifactual loss of labile modifications. Extraction buffers should be optimized to maintain native protein conformation while efficiently solubilizing UMPS from relevant cellular compartments. For studies focusing on specific PTMs, researchers should first immunoprecipitate UMPS using validated antibodies (such as those confirmed effective in HEK-293 cells) followed by Western blot analysis with PTM-specific antibodies . This approach can be complemented by mass spectrometry analysis of immunoprecipitated UMPS to identify novel modification sites. When investigating the functional consequences of PTMs, correlation with enzyme activity assays measuring the conversion of orotic acid to UMP provides important contextual information. Researchers should consider developing site-specific antibodies that recognize UMPS only when modified at particular residues, though such reagents require rigorous validation to confirm specificity. For comprehensive PTM mapping, orthogonal approaches combining antibody-based detection with mass spectrometry provide the most robust results. When studying dynamic changes in UMPS modification, time-course experiments with synchronized cellular perturbations can reveal regulatory mechanisms. Researchers should acknowledge that different isoforms of UMPS (53 kDa, 43 kDa, 33 kDa, and 23 kDa) may exhibit distinct patterns of post-translational modification, necessitating isoform-specific analysis in many cases .

How can UMPS antibodies contribute to translational research bridging basic science and clinical applications?

UMPS antibodies provide valuable tools for translational research connecting fundamental pyrimidine metabolism to clinical applications across multiple disease contexts. In precision oncology, immunohistochemical analysis of UMPS expression in patient tumor samples can potentially predict response to therapies targeting nucleotide metabolism, with human colon cancer tissue being specifically validated for such applications . Correlation of UMPS expression patterns with treatment outcomes in retrospective studies can identify potential biomarker applications, while prospective validation would establish clinical utility. For metabolic disorders involving pyrimidine synthesis, UMPS antibodies enable protein-level confirmation of genetic findings, connecting genotype to biochemical phenotype. Development of companion diagnostics for drugs targeting the pyrimidine synthesis pathway represents another translational application, with UMPS antibodies providing direct evidence of target engagement and pathway modulation. In the context of immunological disorders, where nucleotide metabolism impacts immune cell function, UMPS antibodies can help characterize metabolic differences between normal and pathological immune responses. For neurodegenerative conditions with metabolic components, spatial analysis of UMPS expression in brain tissue may reveal regional vulnerabilities. When developing such translational applications, researchers should establish standardized protocols for sample collection, processing, and analysis to ensure reproducibility across different clinical settings. Critical validation steps include demonstration of antibody specificity in the specific tissue being studied, establishment of clinically relevant cutoff values, and correlation with orthogonal measures of pathway activity .

What are the emerging trends in UMPS antibody applications for research?

The research landscape for UMPS antibodies continues to evolve, with several emerging trends expanding their utility and application scope. Integration of UMPS antibody detection with spatial multi-omics approaches represents a frontier application, wherein antibody-based protein localization is combined with in situ transcriptomics and metabolomics to provide comprehensive, spatially resolved analysis of pyrimidine metabolism regulation. Single-cell applications are rapidly advancing, with flow cytometry and mass cytometry protocols incorporating UMPS antibodies to investigate heterogeneity in metabolic enzyme expression at unprecedented resolution. Technological developments in super-resolution microscopy are enabling detailed subcellular localization studies of UMPS, potentially revealing previously uncharacterized compartmentalization of pyrimidine synthesis machinery. The development of conformation-specific antibodies that distinguish active versus inactive UMPS conformations offers potential for direct assessment of functional status beyond mere protein levels. In the therapeutic domain, antibody-drug conjugates targeting UMPS in cancer cells with upregulated pyrimidine metabolism represent an intriguing possibility, though still in early experimental stages. As multiplexed immunoassay platforms continue to advance, inclusion of UMPS in comprehensive metabolic enzyme panels will enable integrated pathway analysis across diverse experimental systems. Advanced signal amplification techniques, including poly-HRP conjugation strategies and tyramide signal amplification, are continuously improving detection sensitivity for challenging samples with low UMPS expression . Researchers leveraging these emerging applications should maintain rigorous validation standards while adapting established protocols to new technological platforms.