CPS1 Antibody, HRP conjugated

What is CPS1 and what is its biological significance?

Carbamoyl phosphate synthetase 1 (CPS1) is a mitochondrial enzyme with a molecular weight of approximately 165 kDa that catalyzes the synthesis of carbamoyl phosphate from ammonia and bicarbonate. This reaction represents the first committed step of the urea cycle, which is crucial for removing excess ammonia from cells . CPS1 is predominantly expressed in hepatocytes and intestinal epithelial cells, playing a vital role in nitrogen disposal and proper liver metabolism . Deficiency of CPS1 is an autosomal recessive disorder that causes hyperammonemia, highlighting its essential role in human physiology . Recent research has also identified CPS1 as a potential biomarker in various cancers, including hepatocellular carcinoma (HCC), colorectal, gastric, cervical, and pancreatic malignancies .

What advantages do HRP-conjugated antibodies offer in CPS1 detection?

HRP-conjugated CPS1 antibodies provide several methodological advantages for researchers. The horseradish peroxidase conjugation enables direct detection without secondary antibodies, simplifying experimental workflows and reducing non-specific binding issues . These conjugates facilitate sensitive detection in applications such as Western blotting and immunohistochemistry through various visualization methods, including colorimetric and chemiluminescent techniques . HRP-conjugated antibodies typically offer enhanced sensitivity compared to unconjugated antibodies, allowing for detection of low abundance targets with reduced background signals when optimized properly . For CPS1 specific applications, these conjugates have demonstrated efficacy in detecting the protein in multiple species including human, mouse, and rat tissues .

How do monoclonal and polyclonal CPS1 antibodies differ in research applications?

Monoclonal CPS1 antibodies, such as clone EPR7493-3 (ab198969) or OTI7B6, recognize specific epitopes of the CPS1 protein, providing high specificity and reduced background compared to polyclonal alternatives . These antibodies offer consistent lot-to-lot reproducibility, making them ideal for longitudinal studies where experimental conditions must remain standardized . Conversely, polyclonal CPS1 antibodies (e.g., ab45956) recognize multiple epitopes on the target protein, potentially providing higher sensitivity for applications with low abundance targets but with potential for increased cross-reactivity . The choice between monoclonal and polyclonal depends on the specific research requirements - monoclonals excel in applications demanding high specificity like distinguishing hepatocellular carcinomas from other carcinomas, while polyclonals may be preferred when signal amplification is the priority .

What are the validated applications for CPS1-HRP antibodies?

CPS1-HRP antibodies have been validated for several research applications with specific optimization parameters. For Western blotting, these antibodies have demonstrated efficacy at dilutions ranging from 1/5000 (ab198969) to 1/1000, detecting bands at approximately 150-165 kDa in liver tissue and hepatocyte cell lines . In immunohistochemistry on paraffin-embedded sections (IHC-P), optimal dilutions typically range from 1/100 to 1/1000, with heat-mediated antigen retrieval using sodium citrate buffer (pH 6.0) showing good results . CPS1-HRP antibodies work effectively across multiple species, with validated reactivity in human, mouse, and rat samples . Researchers should note that CPS1 is predominantly expressed in liver tissue and hepatocytes, making these the most appropriate positive control samples .

How should I optimize Western blot protocols for CPS1-HRP conjugated antibodies?

For optimal Western blot results with CPS1-HRP antibodies, follow this methodological approach: Begin with sample preparation using a 4-12% Bis-tris gel under the MOPS buffer system, running at 200V for approximately 50 minutes . Transfer proteins onto a nitrocellulose membrane at 30V for 70 minutes, followed by blocking with 2% Bovine Serum Albumin for one hour . Incubate the membrane with diluted CPS1-HRP antibody (1/5000 for ab198969 or similar concentration for other clones) overnight at 4°C . Visualization is best achieved using enhanced chemiluminescence (ECL) development solutions, with high sensitivity substrates recommended for detecting lower expression levels . When analyzing results, expect a band at approximately 150-165 kDa, with potential slight variations depending on post-translational modifications in different tissues . Include appropriate positive controls such as human fetal liver, mouse liver, or rat liver lysates at 10 μg loading concentration .

What troubleshooting approaches should I use for non-specific binding with CPS1-HRP antibodies?

When encountering non-specific binding with CPS1-HRP antibodies, implement these methodological solutions: First, optimize blocking conditions by testing different blocking agents (BSA vs. non-fat milk) and increasing blocking time to 2 hours at room temperature . Consider dilution optimization by testing a concentration gradient for your specific antibody (1/1000 to 1/10,000) to identify the optimal signal-to-noise ratio . Reduce primary antibody incubation temperature to 4°C and extend incubation time to overnight to improve specificity . Incorporate additional washing steps (5 washes of 5 minutes each) with 0.1% Tween-20 in PBS to remove unbound antibodies . For persistent background issues, try adding 0.1-0.5% Triton X-100 to washing buffers to reduce hydrophobic interactions causing non-specific binding . If using tissue samples, ensure proper fixation and antigen retrieval, as overfixation can lead to increased background staining .

How can I optimize immunohistochemistry protocols for CPS1-HRP antibodies?

For optimized immunohistochemistry using CPS1-HRP antibodies, implement this technical approach: Begin with deparaffinization and rehydration of sections following standard protocols . Perform heat-mediated antigen retrieval using sodium citrate buffer (pH 6.0) as recommended for CPS1 detection . Block endogenous peroxidase activity using 3% hydrogen peroxide in methanol for 10 minutes . Apply protein blocking with 1-5% BSA in PBS for 30-60 minutes at room temperature . Dilute CPS1-HRP antibody to 1/1000 (or as recommended for your specific clone) in antibody diluent and incubate overnight at 4°C in a humidified chamber . After thorough washing with PBS-T (5 washes, 5 minutes each), develop signal using DAB substrate kit until optimal staining intensity is achieved (typically 2-10 minutes) . Counterstain with hematoxylin, dehydrate through graded alcohols, clear in xylene, and mount with permanent mounting medium . Include positive controls (normal liver tissue) and negative controls (omission of primary antibody) in each experiment .

How can CPS1-HRP antibodies be used to differentiate hepatocellular carcinomas from other carcinomas?

CPS1-HRP antibodies serve as powerful diagnostic tools in differentiating hepatocellular carcinomas (HCCs) from other carcinomas through several methodological approaches. CPS1 is a hepatocyte-specific marker that localizes to mitochondria of hepatocytes, making it highly specific for liver-derived tissues . In immunohistochemical applications, use dilutions of 1/1000 for HRP-conjugated antibodies on formalin-fixed, paraffin-embedded tissue sections with heat-mediated antigen retrieval . Positive CPS1 staining presents as strong cytoplasmic signal in hepatocytes and HCC cells, while being absent in most other carcinoma types, including cholangiocarcinomas . This staining pattern allows pathologists to distinguish primary HCCs from metastatic tumors to the liver with high sensitivity and specificity . Research indicates that strong CPS1 expression correlates with smaller tumor size and improved patient survival in HCC cases, providing prognostic value beyond mere diagnosis . For challenging cases, consider using CPS1 in a panel with other hepatocyte markers such as Hep Par 1 for increased diagnostic accuracy .

What considerations are important when studying CPS1 expression in urea cycle disorders?

When studying CPS1 expression in urea cycle disorders using HRP-conjugated antibodies, researchers should implement several critical methodological considerations. First, ensure appropriate sampling by obtaining liver biopsies when possible, as CPS1 is predominantly expressed in hepatocytes, with careful handling to preserve protein integrity . Use Western blotting with CPS1-HRP antibodies at 1/5000 dilution to quantitatively assess protein expression levels, always comparing against age and sex-matched control samples . For immunohistochemical analysis, implement heat-mediated antigen retrieval with sodium citrate buffer (pH 6.0) and use antibody dilutions of 1/1000, examining not only presence/absence but also subcellular localization . Analyze results in context of patient's clinical presentation and ammonia levels, as CPS1 deficiency manifests as hyperammonemia . Consider genetic analysis alongside protein expression studies, as various mutations can affect protein expression, stability, and activity differently . For comprehensive analysis, measure enzyme activity in concert with protein expression, as some mutations may produce immunoreactive but enzymatically defective protein .

How does CPS1 expression vary across different cancer types and what implications does this have for antibody selection?

CPS1 expression demonstrates significant heterogeneity across different cancer types, necessitating careful antibody selection and experimental design. In hepatocellular carcinomas (HCCs), CPS1 is typically strongly expressed, making it a valuable diagnostic marker when using HRP-conjugated antibodies at 1/1000 dilution in IHC applications . Research shows variable CPS1 expression in gastric carcinomas, particularly those with intestinal metaplasia, requiring more sensitive detection methods such as signal amplification or higher antibody concentrations (1/500) . In colorectal, cervical, and pancreatic cancers, CPS1 expression has been detected but at generally lower levels compared to HCCs, necessitating optimized protocols with overnight primary antibody incubation at 4°C . Interestingly, strong CPS1 expression has been correlated with smaller tumor size and longer patient survival in some cancers, suggesting its potential value as a prognostic biomarker . When studying non-hepatic tumors, researchers should select highly sensitive detection systems and include appropriate positive (liver) and negative controls to ensure accurate interpretation of results .

What validation steps are essential before using CPS1-HRP antibodies in a new experimental system?

Before implementing CPS1-HRP antibodies in a new experimental system, researchers should conduct these essential validation steps: First, perform positive control testing using tissues or cells known to express CPS1 (human liver, HepG2 cells) to verify antibody functionality . Run negative control analyses with tissues known not to express CPS1 or with CPS1-knockout models to confirm specificity . Conduct epitope analysis by reviewing the antibody's immunogen sequence for potential cross-reactivity with other proteins in your experimental system . For Western blotting, verify the molecular weight (expected 150-165 kDa) and band pattern across different sample types . Perform dilution series experiments (1/500, 1/1000, 1/5000) to determine optimal concentration for your specific samples and application . Compare results from at least two different CPS1 antibodies (ideally recognizing different epitopes) to confirm findings . For IHC applications, run peptide competition assays where available to confirm staining specificity . Document batch-to-batch consistency by recording lot numbers and standardizing experimental conditions for longitudinal studies .

How can I preserve CPS1-HRP antibody activity during long-term storage?

To maintain optimal activity of CPS1-HRP conjugated antibodies during long-term storage, implement these evidence-based preservation strategies: Store antibodies at -20°C in a frost-free freezer as recommended by manufacturers, with glycerol-based storage buffers (50% glycerol) to prevent freeze-thaw damage . Aliquot the antibody into single-use volumes (10-20 μL) immediately upon receipt to minimize repeated freeze-thaw cycles, as each cycle can significantly reduce HRP enzymatic activity . When handling, always keep antibodies on ice or at 4°C during experiments and avoid exposing to room temperature for extended periods . Reconstituted or diluted antibodies should be supplemented with stabilizing proteins (1% BSA) and preservatives (0.09% sodium azide) to prevent microbial contamination and protein degradation . Before using stored antibodies, centrifuge vials briefly to collect contents at the bottom and avoid pipetting directly from the surface where protein aggregation may occur . Monitor antibody performance over time by including standardized positive controls in each experiment and tracking signal intensity changes . If reduced activity is observed after storage, consider using signal enhancement methods such as tyramide signal amplification to compensate for potential loss of HRP activity .

What antigen retrieval methods work best for CPS1 detection in fixed tissues?

For optimal CPS1 detection in fixed tissues, heat-mediated antigen retrieval methods have demonstrated superior results compared to enzymatic approaches. The most effective protocol involves using sodium citrate buffer (pH 6.0) in a pressure-controlled environment, such as a pressure cooker or retrieval module at 95-98°C for 20 minutes . This approach effectively breaks protein cross-links formed during fixation without damaging tissue morphology or epitope structure . For highly fixed samples, extending retrieval time to 30 minutes may improve antigen accessibility . EDTA-based buffers (pH 8.0-9.0) have shown variable results with CPS1 antibodies and should be tested in parallel with citrate buffers to determine optimal conditions for specific sample types . Importantly, consistent retrieval parameters (time, temperature, buffer composition) must be maintained across experimental samples to ensure comparable staining results . After retrieval, allowing sections to cool gradually to room temperature for 20 minutes prevents tissue detachment from slides and reduces background staining . For automated staining platforms, manufacturers' recommended retrieval solutions compatible with CPS1 antibodies should be followed, with proper validation using positive control tissues (human liver sections) .

How can I quantify CPS1 expression levels using HRP-conjugated antibodies?

For accurate quantification of CPS1 expression using HRP-conjugated antibodies, implement these methodological approaches: In Western blot applications, use a dilution series of recombinant CPS1 protein (10-100 ng) alongside samples to create a standard curve for relative quantification . Include housekeeping protein controls (β-actin, GAPDH) for normalization, and use image analysis software (ImageJ, Bio-Rad Image Lab) to measure band intensity in the linear detection range . For immunohistochemistry quantification, employ computer-assisted image analysis using platforms like QuPath or ImageJ with color deconvolution to separate DAB (CPS1) signal from hematoxylin counterstain . Score staining intensity on a scale (0-3+) and calculate H-scores (0-300) by multiplying intensity by percentage of positive cells for semi-quantitative analysis . For more precise quantification, consider using specialized ELISA techniques with HRP-conjugated CPS1 antibodies, measuring absorbance with spectrophotometric plate readers and comparing to standard curves . When comparing expression across different experimental conditions, always process samples simultaneously using identical antibody lots, incubation times, and development conditions to minimize technical variability . For longitudinal studies, include internal reference samples with known CPS1 expression in each experiment to control for batch effects .

What control samples are essential when working with CPS1-HRP antibodies?

When working with CPS1-HRP antibodies, a comprehensive control strategy is essential for experimental validity. Primary positive controls should include human liver tissue or hepatocyte-derived cell lines (HepG2), where CPS1 is abundantly expressed . Include mouse and rat liver samples when working across species, as CPS1 is conserved but may show species-specific patterns . Negative control tissues should include non-hepatic tissues such as muscle or lung where CPS1 expression is minimal or absent . Technical negative controls should omit primary antibody while maintaining all other reagents and steps to identify non-specific binding of detection systems . For antibody specificity validation, include peptide competition controls where available, pre-incubating the antibody with immunizing peptide to confirm signal specificity . When possible, include samples from CPS1-deficient patients or knockout models as definitive negative controls, though these may be difficult to obtain . For Western blotting, molecular weight markers must be included to confirm the expected 165 kDa band size of CPS1 . Loading controls (β-actin, GAPDH) are essential for normalization and ensuring equal protein loading across samples .

How can multiplex immunostaining be achieved with CPS1-HRP antibodies?

For successful multiplex immunostaining incorporating CPS1-HRP antibodies, implement this sequential methodology: Begin with the CPS1-HRP antibody at 1/1000 dilution as the first staining layer, developing with tyramide-based signal amplification systems that covalently deposit fluorophores onto the tissue . After development, perform complete antibody stripping using glycine buffer (pH 2.2) or commercial antibody removal solutions to eliminate the HRP activity while preserving the deposited fluorophore . Apply subsequent antibodies targeting different proteins, ensuring they are raised in different host species than the CPS1 antibody to prevent cross-reactivity . For chromogenic multiplexing, use different substrates for each antibody (DAB for brown, AEC for red, etc.) in sequential staining rounds with complete HRP inactivation between cycles using 3% hydrogen peroxide . When designing multiplex panels, pair CPS1 with complementary markers such as CK19 (for bile ducts) or CD68 (for Kupffer cells) to provide comprehensive tissue analysis . Microscopic analysis should include multispectral imaging systems capable of separating closely overlapping chromogens or fluorophores for accurate co-localization studies . Always include single-stained control slides for each antibody to establish proper exposure settings and confirm the absence of crosstalk between detection channels .

How are CPS1-HRP antibodies used in studying liver pathologies beyond cancer?

CPS1-HRP antibodies provide valuable tools for investigating various liver pathologies through multiple methodological approaches. In hepatitis C (HCV) research, these antibodies at 1/1000 dilution can assess CPS1 expression changes in infected hepatocytes, as altered urea cycle function has been implicated in disease progression . For studying hepatic fibrosis, researchers use CPS1-HRP antibodies in dual immunostaining protocols alongside markers of stellate cell activation (α-SMA) to investigate the relationship between functional hepatocyte loss and fibrogenic activity . In fatty liver disease models, Western blotting with CPS1-HRP antibodies (1/5000 dilution) helps quantify hepatocyte-specific protein changes in response to steatosis and steatohepatitis, correlating expression with metabolic parameters . For developmental studies, these antibodies enable tracking of hepatocyte maturation through CPS1 expression patterns in embryonic and neonatal tissues . In transplantation research, CPS1 immunostaining serves as a marker of functional hepatocyte engraftment, with quantitative analysis possible through computer-assisted image analysis of staining intensity and distribution . For toxicological investigations, changes in CPS1 expression monitored via Western blotting or IHC provide insights into hepatocyte-specific damage from various compounds .

What insights has CPS1 research provided into cancer metabolism?

Recent research utilizing CPS1-HRP antibodies has revealed significant insights into cancer metabolism and the potential therapeutic implications of CPS1 in various malignancies. Studies have shown that CPS1 expression correlates with tumor size and patient survival in hepatocellular carcinoma (HCC), with stronger expression associated with better prognosis . This suggests CPS1's potential role as both a diagnostic and prognostic marker. Western blot analysis using HRP-conjugated CPS1 antibodies has demonstrated differential expression patterns across cancer cell lines, revealing metabolic adaptations specific to the tissue of origin . In liver cancer, CPS1 appears to regulate the balance between ureagenesis and pyrimidine synthesis, potentially influencing nucleotide availability for rapidly dividing cells . Interestingly, while CPS1 is predominantly expressed in liver, its expression has been detected in various non-hepatic tumors including gastric, colorectal, cervical, and pancreatic cancers, suggesting cancer-specific metabolic reprogramming . Mechanistic studies indicate that CPS1 may contribute to alternative nitrogen metabolism pathways in cancer cells, potentially providing novel therapeutic targets . The variability in CPS1 expression across different tumor types highlights the complexity of cancer metabolism and underscores the importance of personalized approaches to cancer therapy based on metabolic profiles .