CSP1 Antibody

What is CPS1 and what is its primary biological function?

CPS1 (Carbamoyl Phosphate Synthetase 1) is a mitochondrial enzyme that catalyzes the first and rate-limiting step of the urea cycle, converting ammonia to carbamoyl phosphate. This critical enzyme plays an essential role in nitrogen metabolism and ammonia detoxification, primarily in the liver. CPS1 functions to maintain nitrogen balance in the body by facilitating the removal of excess nitrogen resulting from protein metabolism . The enzyme contains 1500 amino acids with a calculated molecular weight of 165 kDa, though it may be observed between 140-165 kDa in experimental contexts .

The enzyme's functionality is particularly important in hepatocytes, where disruption of CPS1 activity can lead to hyperammonemia and related metabolic disorders. These conditions highlight CPS1's integral role in nitrogen homeostasis and cellular metabolism .

How is CPS1 expression regulated in normal tissues versus pathological conditions?

In normal physiology, CPS1 expression is predominantly liver-specific, with robust expression in hepatocytes. Recent research has revealed that CPS1 expression follows a distinctive pattern in various pathological conditions:

• In Correa's cascade (progression to gastric cancer): CPS1 shows diffuse and strongly positive expression (100% of cases) in intestinal metaplasia of the glandular epithelium, but gradually decreases as lesions progress toward malignancy .

• In hepatocellular carcinoma (HCC): CPS1 expression is frequently suppressed at the transcriptional level compared to normal hepatocytes .

• In cancer biology: Hypermethylation-mediated downregulation of CPS1 expression may contribute to the progression from normal hepatocytes to HCC .

Interestingly, tumor suppressor p53 has been found to repress the expression of urea cycle enzymes including CPS1, OTC, and ARG1, causing ammonia accumulation which suppresses cancer growth .

What criteria should researchers consider when selecting a CPS1 antibody for specific applications?

Selecting the appropriate CPS1 antibody requires careful consideration of several experimental parameters:

• Host species and antibody type: Commercial CPS1 antibodies are commonly raised in rabbits as polyclonal antibodies (e.g., CAB8080, #84510, 24513-1-AP) . Polyclonal antibodies offer high sensitivity but may introduce batch-to-batch variability.

• Species reactivity: Verify cross-reactivity with your experimental model. Available antibodies show different reactivity profiles:

• Application compatibility: Select antibodies validated for your specific application. Different antibodies perform optimally in different contexts:

• Target epitope: Consider the specific epitope recognized by the antibody. For example, CAB8080 targets a sequence corresponding to amino acids 750-970 of human CPS1 .

What validation steps are essential before using a CPS1 antibody in critical research?

Before employing a CPS1 antibody in pivotal experiments, researchers should implement a comprehensive validation strategy:

• Positive control selection: Use tissues/cells known to express CPS1 at high levels. Validated positive controls include:

  • HepG2 cells, HeLa cells, L02 cells (human)

  • Liver tissue (mouse and rat)

• Negative control implementation: Include tissues/cells with minimal CPS1 expression or use CPS1 knockdown/knockout models.

• Specificity verification:

  • Western blot analysis should show a single band at the expected molecular weight (140-165 kDa)

  • For IHC/IF, include peptide competition assays to confirm binding specificity

  • Cross-reference expression patterns with established data on CPS1 tissue distribution

• Technical optimization:

  • For IHC, test different antigen retrieval methods (e.g., TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Titrate antibody concentrations to determine optimal signal-to-noise ratio

  • Validate secondary antibody specificity

• Correlation with functional assays: Where possible, correlate antibody-based detection with functional assays of CPS1 enzymatic activity.

How can researchers effectively use CPS1 antibodies to investigate urea cycle disorders?

Investigating urea cycle disorders with CPS1 antibodies requires sophisticated experimental approaches:

• Patient-derived samples analysis:

  • Use Western blotting to quantify CPS1 protein levels in liver biopsies from patients with suspected CPS1 deficiency

  • Implement IHC to visualize CPS1 distribution patterns in affected tissues

  • Compare expression levels between patients and healthy controls using standardized immunoblotting protocols

• Mutation impact assessment:

  • Generate expression constructs containing patient-specific CPS1 mutations

  • Transfect appropriate cell lines and use antibodies to assess protein expression, stability, and subcellular localization

  • Correlate antibody-detected expression levels with enzyme activity assays

• Regulatory mechanism investigation:

  • Use chromatin immunoprecipitation followed by CPS1 antibody detection to identify transcriptional regulators

  • Employ pulse-chase experiments with CPS1 antibodies to determine protein half-life in normal versus pathological conditions

  • Investigate post-translational modifications using modification-specific antibodies alongside general CPS1 antibodies

• Therapeutic response monitoring:

  • Utilize CPS1 antibodies to track protein expression changes in response to experimental treatments

  • Develop quantitative IHC protocols with standardized scoring systems similar to that described for gastric cancer research

What are the optimal protocols for using CPS1 antibodies in cancer research?

CPS1 has emerged as an important factor in cancer biology, with specific protocols optimized for oncology research:

• Expression profiling in tumors:

  • For IHC analysis of tumor tissues, implement a standardized scoring system:

    • Staining intensity: No staining (0), faint (1), moderate (2), dark (3)

    • Percentage of positive cells: No positive cells (0), ≤25% (1), 25–49% (2), 50–75% (3), >75% (4)

    • Calculate immunoreactive score (IRS) as intensity × percentage

    • Categorize as: Strong positive (IRS 8-12), focal positive (IRS 4-6), weak positive (IRS 2-3), negative (IRS 0-1)

• Metabolic reprogramming studies:

  • Use CPS1 antibodies in conjunction with metabolomics to investigate the relationship between CPS1 expression and altered metabolism in cancer cells

  • Employ immunoprecipitation with CPS1 antibodies followed by mass spectrometry to identify interaction partners in cancer-specific contexts

  • Correlate CPS1 expression with pyrimidine/purine ratios in KRAS-mutant cancers

• Therapeutic targeting assessment:

  • Monitor CPS1 expression changes in response to treatments targeting metabolic vulnerabilities

  • Use CPS1 antibodies to evaluate the efficacy of siRNA or small molecule inhibitors in suppressing CPS1 expression or function

  • Implement multiplexed immunofluorescence to simultaneously assess CPS1 and markers of DNA damage following CPS1 targeting

• Prognostic biomarker development:

  • Standardize IHC protocols across laboratories to enable comparative studies of CPS1 as a prognostic indicator

  • Employ tissue microarrays with CPS1 antibodies to efficiently screen large cohorts

  • Correlate CPS1 expression patterns with patient outcomes, tumor stage, and treatment response

What are common issues encountered with CPS1 antibodies and how can they be resolved?

Researchers frequently encounter several challenges when working with CPS1 antibodies:

• High background in immunohistochemistry:

  • Solution: Optimize blocking conditions using 5-10% normal serum from the same species as the secondary antibody

  • Solution: Increase washing duration and frequency between steps

  • Solution: Titrate primary antibody concentration (starting with 1:50-1:500 for IHC)

• Weak or absent signal in Western blot:

  • Solution: Ensure adequate protein loading (50-100 μg total protein)

  • Solution: Optimize transfer conditions for high molecular weight proteins (165 kDa)

  • Solution: Use gradient gels (4-15%) to improve resolution of large proteins

  • Solution: Adjust antibody concentration (try 1:500-1:1000)

• Multiple bands in Western blot:

  • Solution: Verify sample preparation to minimize protein degradation (use fresh protease inhibitors)

  • Solution: Test different lysis buffers to preserve protein integrity

  • Solution: Perform peptide competition assays to identify specific bands

• Inconsistent staining patterns:

  • Solution: Standardize fixation protocols (fixation time and conditions significantly impact epitope accessibility)

  • Solution: Test multiple antigen retrieval methods (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

  • Solution: Ensure consistent antibody handling and storage (avoid repeated freeze-thaw cycles)

How should researchers design experiments to address contradictory findings regarding CPS1 expression in different cancer types?

Addressing contradictory findings about CPS1 in cancer research requires systematic experimental approaches:

• Comprehensive molecular profiling:

  • Design experiments that simultaneously assess CPS1 at genomic, transcriptomic, and proteomic levels

  • Implement bisulfite sequencing to evaluate CPS1 promoter methylation status alongside expression analysis

  • Correlate findings with chromosomal alterations and mutational profiles

• Context-dependent expression analysis:

  • Design tissue microarrays representing multiple cancer types, stages, and grades

  • Employ multiplexed immunofluorescence to simultaneously visualize CPS1 with lineage markers and key oncogenic drivers

  • Analyze expression patterns in relation to tumor microenvironment features

• Functional validation approaches:

  • Implement CRISPR-Cas9 gene editing to create isogenic cell lines with CPS1 knockout

  • Perform rescue experiments with wild-type and mutant CPS1 constructs

  • Assess phenotypic consequences through proliferation, migration, and metabolic assays

• Reconciliation framework for contradictory findings:

  • Document all experimental variables that could influence results (antibody clones, detection methods, scoring systems)

  • Create standardized protocols for CPS1 detection across research groups

  • Develop an integrated analysis approach that considers cancer-specific metabolic contexts

How do researchers distinguish between applications of CPS1 antibodies and CSP antibodies?

It is crucial for researchers to clearly distinguish between CPS1 (Carbamoyl Phosphate Synthetase 1) and CSP (Circumsporozoite Protein) antibodies, as they target entirely different proteins with distinct research applications:

• Target protein differences:

  • CPS1 antibodies: Target a human/mammalian mitochondrial enzyme involved in the urea cycle

  • CSP antibodies: Target a Plasmodium falciparum surface protein involved in malaria parasite infection

• Research context:

  • CPS1 research: Primarily focuses on metabolic disorders, liver function, and certain cancers

  • CSP research: Exclusively relates to malaria parasitology and vaccine development

• Experimental applications:

  • When investigating malaria protection mechanisms, researchers should specifically use anti-CSP antibodies that target epitopes such as the (NPNA)n repeat region

  • For urea cycle or hepatocellular studies, CPS1-specific antibodies are appropriate

What methodological approaches optimize the use of CSP antibodies in malaria research?

For researchers working with CSP antibodies in malaria studies:

• Protection assays:

  • The WRAIR intravenous challenge model can be used to assess protection, measuring the ability of CSP antibodies to neutralize 100% of parasites in the blood

  • Consider technical variability between challenge experiments, as protection outcomes can vary (e.g., between 24 and 48h challenges)

• Epitope targeting optimization:

  • Compare high-affinity (NPNA)n-specific mAbs with dual-specific mAbs like CIS43

  • mAb 317 has shown consistently higher protection (80-100%) compared to CIS43 (40-50%) across independent challenges

• Assay prioritization:

  • Prioritize affinity and avidity assays on Fab and bivalent mAbs

  • Implement ILSDA (inhibition of liver-stage development assay) alongside in vivo challenge models

  • Consider these assays essential for down-selection of future CSP-based interventions

How might emerging technologies enhance the utility of CPS1 antibodies in research?

Emerging technologies promise to revolutionize CPS1 antibody applications:

• Single-cell proteomics integration:

  • Combine CPS1 antibodies with mass cytometry (CyTOF) to analyze CPS1 expression at single-cell resolution

  • Integrate with spatial transcriptomics to correlate protein expression with gene expression patterns in tissue context

  • Develop multiplexed imaging approaches to simultaneously visualize CPS1 with multiple metabolic enzymes

• Advanced structural and functional analysis:

  • Employ super-resolution microscopy with CPS1 antibodies to visualize subcellular localization at nanoscale resolution

  • Develop proximity labeling approaches using CPS1 antibodies to identify novel interaction partners

  • Create optogenetic tools coupled with CPS1 antibody detection for dynamic monitoring of enzyme regulation

• Therapeutic development platforms:

  • Utilize CPS1 antibodies in high-content screening assays to identify modulators of expression or activity

  • Develop antibody-drug conjugates targeting CPS1-expressing cells in specific disease contexts

  • Create biosensor systems incorporating CPS1 antibody fragments to monitor enzyme activity in real-time

What are the potential applications of CPS1 antibodies in precision medicine?

CPS1 antibodies may contribute significantly to precision medicine approaches:

• Diagnostic stratification:

  • Develop standardized IHC protocols for CPS1 detection in tumor biopsies to guide treatment decisions

  • Create multiplex IHC panels combining CPS1 with other metabolic markers to classify tumors based on metabolic phenotypes

  • Integrate CPS1 expression data with genomic profiling to identify patient subgroups most likely to benefit from metabolism-targeted therapies

• Treatment response monitoring:

  • Use serial liquid biopsies with circulating tumor cell CPS1 analysis to track treatment efficacy

  • Develop imaging approaches using radiolabeled CPS1 antibodies for non-invasive monitoring

  • Create companion diagnostic assays based on CPS1 expression to guide patient selection for clinical trials

• Novel therapeutic approaches:

  • Design immunotherapy strategies targeting CPS1-expressing cells in specific cancer contexts

  • Develop CPS1 inhibitors for cancers that show dependence on CPS1 function, such as those with KRAS mutations and LKB1 loss

  • Create synthetic lethal approaches exploiting metabolic vulnerabilities in cells with altered CPS1 expression