SERPINC1 Antibody, HRP conjugated

What is SERPINC1 and why is it important in research?

SERPINC1 (serpin family C member 1) encodes antithrombin-III, the most important serine protease inhibitor in plasma that regulates the blood coagulation cascade. It inhibits thrombin, matriptase-3/TMPRSS7, and factors IXa, Xa, and XIa, with its inhibitory activity greatly enhanced in the presence of heparin . Beyond its classical role in coagulation, SERPINC1 has emerged as a potential tumor suppressor in hepatocellular carcinoma, where it induces apoptosis in cancer cells and regulates tumor immunity . This dual role makes SERPINC1 an important target for research in both hematology and oncology fields.

How does the HRP conjugation in SERPINC1 antibody enhance research applications?

The horseradish peroxidase (HRP) conjugation to SERPINC1 antibody provides significant methodological advantages for researchers. The direct conjugation eliminates the need for secondary antibodies in ELISA applications, reducing background signal and potential cross-reactivity issues . HRP generates a colorimetric reaction that offers excellent sensitivity for quantitative detection of antithrombin-III in complex biological samples. This conjugation enables more streamlined experimental workflows, particularly in sandwich ELISA techniques where the antibody can directly bind to the target protein and produce a measurable signal without additional amplification steps .

What are the known structural features of SERPINC1 that researchers should consider when using this antibody?

When designing experiments with SERPINC1 antibody, researchers should account for the specific epitope targeting (amino acids 37-180 of human antithrombin-III) . This region may influence binding efficiency in various applications. SERPINC1 belongs to the serpin superfamily characterized by a specific tertiary structure including a reactive center loop. Mutations in this region can significantly alter protein function, as evidenced in hereditary antithrombin deficiency studies . The antibody's polyclonal nature means it recognizes multiple epitopes within this region, providing robust detection capability but potentially variable specificity compared to monoclonal alternatives . Understanding these structural features is critical when interpreting experimental results, particularly when studying mutant forms or protein-protein interactions.

What are the optimal storage conditions for maintaining SERPINC1 antibody activity, and how can researchers validate antibody functionality before critical experiments?

SERPINC1 antibody, HRP conjugated, should be stored at -20°C or -80°C upon receipt, with repeated freeze-thaw cycles strictly avoided to maintain conjugate stability . For long-term storage planning, the antibody remains stable at these temperatures for approximately 12 months. Before conducting critical experiments, researchers should validate antibody functionality through positive control testing using samples with known SERPINC1 expression levels. A small-scale ELISA with serially diluted antibody concentrations (typically 1:1000 to 1:10000) can establish the optimal working concentration and confirm signal-to-noise ratios. Additionally, examining the preservation of the liquid state (without visible precipitates) and normal coloration provides a quick assessment of potential denaturation. For rigorous validation, researchers can compare results against alternative detection methods such as Western blotting with unconjugated antibodies or mass spectrometry-based validation.

What methodological approaches can researchers use to study SERPINC1's dual role in coagulation and tumor suppression simultaneously?

To investigate SERPINC1's dual functionality, researchers should employ integrated experimental designs that capture both coagulation regulation and tumor suppression properties. A comprehensive approach would include:

• Functional activity assays: Measuring anti-FXa and anti-thrombin activities using chromogenic substrates to assess coagulation inhibition capacity

• Cell-based assays: Employing cell lines (such as HepG2 and SMMC7721) with SERPINC1 overexpression or knockdown to evaluate:

  • Apoptosis induction (through Annexin V/PI staining, caspase activity assays)

  • Cell proliferation inhibition (MTT/CCK-8 assays)

  • Macrophage polarization effects through co-culture systems with THP1 cells

• Molecular signaling analysis: Investigating ubiquitination patterns and protein degradation pathways affected by SERPINC1, focusing on apoptosis, autophagy, and VEGF signaling molecules

• Tumor microenvironment studies: Analyzing immune cell infiltration and polarization markers (CD163 for M2 macrophages and CD80 for M1 macrophages) in response to SERPINC1 modulation

This integrated approach allows researchers to establish mechanistic links between SERPINC1's anticoagulant properties and its role in cancer immunomodulation.

How can researchers optimize SERPINC1 antibody concentration for ELISA assays across different sample types?

Optimizing SERPINC1 antibody concentration requires systematic titration experiments tailored to specific sample types. For standard ELISA optimization:

• Initial range-finding: Perform a broad titration (1:500 to 1:10,000) using positive control samples with known SERPINC1 expression

• Sample-specific optimization:

  • Serum/plasma samples: Start with higher dilutions (1:2000-1:5000) due to naturally high SERPINC1 expression

  • Cell culture supernatants: Lower dilutions (1:500-1:2000) may be required, depending on cell type

  • Tissue homogenates: Require optimization based on tissue origin and preparation method

• Signal-to-noise assessment: Calculate signal-to-background ratios at each concentration; optimal dilution typically yields ratio >10:1

• Standard curve validation: At the selected antibody concentration, verify linearity (R² > 0.98) across the expected concentration range

• Cross-validation: Compare results with alternative quantification methods (e.g., immunoblotting)

For challenging samples with potentially interfering substances, researchers should consider adding blocking agents (1-5% BSA) to reduce background and implementing more stringent washing steps (5-6 washes with 0.05% Tween-20) .

How does SERPINC1 expression correlate with hepatocellular carcinoma progression, and what methodological approaches best capture this relationship?

SERPINC1 expression demonstrates a complex relationship with hepatocellular carcinoma (HCC) progression. Research shows that while SERPINC1 is upregulated in HCC compared to normal tissues, its expression is negatively correlated with advancing tumor grades and stages . This paradoxical pattern suggests an early compensatory upregulation followed by progressive loss with disease advancement.

Methodologically, researchers should employ multi-modal approaches to comprehensively assess this relationship:

• Transcriptomic analysis: RNA-seq or qPCR to quantify SERPINC1 mRNA levels across different tumor grades

• Protein expression analysis: Immunohistochemistry with semi-quantitative scoring systems (0-3+ intensity scale) to evaluate protein expression patterns in tissue microarrays containing multiple tumor grades and matched normal tissues

• Clinicopathological correlation: Statistical analysis correlating SERPINC1 expression with:

  • Tumor grade/stage (TNM classification)

  • Patient survival data (Kaplan-Meier analysis)

  • Tumor immune microenvironment metrics

• Functional validation: In vitro studies using cell lines representing different HCC progression stages to verify phenotypic effects of SERPINC1 modulation

This comprehensive approach has revealed that higher SERPINC1 expression correlates with better prognosis and improved tumor immune microenvironment, characterized by decreased M2 macrophage polarization and enhanced anti-tumor immunity .

What is the significance of SERPINC1 mutations in thrombotic disorders, and how can researchers effectively study these mutations?

SERPINC1 mutations represent the primary cause of hereditary antithrombin deficiency, an autosomal dominant thrombotic disorder primarily manifesting as venous thrombosis and pulmonary embolism . These mutations disrupt the critical anticoagulant function of antithrombin-III, leading to hypercoagulable states.

For effective mutation analysis, researchers should implement:

• Genetic screening approaches:

  • Next-generation sequencing targeting SERPINC1 and related genes (PROS1, PROC, HABP2, MTHFR)

  • Sanger sequencing for family segregation analysis and confirmation

• Functional characterization:

  • Expression plasmid construction for wild-type and mutant SERPINC1

  • Transfection into cellular models (e.g., HEK293T cells) to assess:

    • Protein expression levels

    • Cellular localization using immunofluorescence

    • Secretion efficiency through analysis of culture media

• Structural analysis:

  • Bioinformatic modeling of mutation effects on protein structure

  • Assessment of conformational changes and potential impacts on functional domains

• Clinical correlation:

  • Antithrombin activity assays (chromogenic)

  • Antithrombin antigen level measurement

  • Detailed thrombotic event documentation in patient cohorts

This comprehensive approach enables researchers to establish clear genotype-phenotype correlations and elucidate mechanisms by which specific mutations affect antithrombin function and thrombosis risk.

How can researchers leverage SERPINC1 antibodies to investigate the protein's role in macrophage polarization and tumor immunity?

SERPINC1 has emerged as a regulator of tumor immunity through its influence on macrophage polarization, particularly inhibiting the formation of tumor-promoting M2 macrophages . To investigate this function, researchers can implement sophisticated experimental designs:

• Co-culture systems: Establish co-culture models between SERPINC1-manipulated cancer cells (overexpression/knockdown) and monocytes/macrophages (THP1 cells) to directly assess polarization effects, analyzing:

  • M2 marker expression (CD163) and M1 marker expression (CD80) via flow cytometry

  • Expression of M2-associated cytokines (IL4, IL10, IL13) using ELISA with SERPINC1 antibody

• Mechanistic dissection: Employ the SERPINC1 antibody in chromatin immunoprecipitation (ChIP) experiments to identify transcriptional regulation of macrophage polarization genes, coupled with ubiquitinome analysis to examine post-translational regulation of key molecules like HIF1A

• In vivo tumor models: Create SERPINC1-overexpressing tumor xenografts to analyze tumor-associated macrophage infiltration and phenotype in the microenvironment, using the antibody for immunohistochemical analysis

• Multi-parametric flow cytometry: Develop panels to simultaneously assess multiple immune cell populations (macrophages, NK cells, T cells) in response to SERPINC1 modulation, correlating results with tumor progression metrics

• Single-cell RNA sequencing: Apply this technique to tumor samples with varying SERPINC1 expression levels to identify cell-specific transcriptional profiles and communication networks between cancer cells and immune cells

These approaches collectively provide a comprehensive understanding of how SERPINC1 modulates the tumor immune microenvironment through macrophage polarization and other immune mechanisms.

What techniques can researchers use to investigate the relationship between SERPINC1's ubiquitination regulation and its tumor suppressive functions?

SERPINC1's role in regulating ubiquitination processes represents an advanced research frontier with significant implications for its tumor suppressive functions . Researchers can employ several sophisticated techniques to investigate this relationship:

• Ubiquitinome analysis:

  • Tandem ubiquitin binding entity (TUBE) pulldown followed by mass spectrometry

  • Targeted analysis of poly-ubiquitination patterns of proteins in pathways regulated by SERPINC1 (autophagy, apoptosis, lactate metabolism, VEGF signaling)

• Substrate identification:

  • Proximity-based labeling techniques (BioID, APEX) with SERPINC1 as bait

  • Co-immunoprecipitation with SERPINC1 antibody followed by ubiquitin-specific detection

• Functional validation:

  • Proteasome inhibition studies (MG132 treatment) to assess protein stabilization

  • Site-directed mutagenesis of key ubiquitination sites in candidate substrates

  • Deubiquitinase inhibitor treatments to analyze pathway dependency

• Mechanistic dissection:

  • Investigation of specific E3 ligases and deubiquitinases interacting with SERPINC1

  • CRISPR-based screens to identify essential components of the SERPINC1-dependent ubiquitin-proteasome system

• Phenotypic correlation:

  • Correlation between ubiquitination patterns and cellular outcomes (apoptosis, macrophage polarization)

  • Analysis of ubiquitination changes in response to SERPINC1-overexpression in cancer cells

This multifaceted approach will help elucidate how SERPINC1 regulates the ubiquitin-proteasome system to control apoptosis and antitumor immunity, potentially revealing novel therapeutic targets at the intersection of these pathways .

What are the common sources of experimental variability when using SERPINC1 antibody in ELISA, and how can researchers systematically address them?

When using SERPINC1 antibody in ELISA applications, researchers may encounter several sources of variability that can affect data reliability. These challenges and their systematic solutions include:

• Antibody degradation issues:

  • Problem: Activity loss due to improper storage or handling

  • Solution: Store at recommended temperatures (-20°C or -80°C), avoid repeated freeze-thaw cycles, prepare single-use aliquots, and validate each lot before critical experiments

• Sample matrix interference:

  • Problem: Components in serum, plasma, or tissue homogenates may affect antibody binding

  • Solution: Optimize sample dilution factors (typically 1:10 to 1:100), use appropriate blocking buffers (5% BSA), and include matrix-matched standards and controls

• Cross-reactivity concerns:

  • Problem: Potential binding to related serpins or non-specific interactions

  • Solution: Validate specificity using positive and negative control samples, consider competitive blocking with recombinant SERPINC1, and verify results with alternative detection methods

• Inter-assay variability:

  • Problem: Inconsistent results between experimental runs

  • Solution: Include standard curves on each plate, utilize consistent reference samples across experiments, and normalize data to appropriate housekeeping proteins or total protein content

• Detection limit challenges:

  • Problem: Insufficient sensitivity for low-expressing samples

  • Solution: Optimize antibody concentration through titration experiments, extend substrate incubation time (while monitoring background), and consider signal amplification systems

Implementing these systematic approaches ensures more reliable and reproducible results when working with SERPINC1 antibody in quantitative applications, particularly for comparative studies across different experimental conditions or sample types.

How can researchers validate SERPINC1 antibody specificity for studying mutant variants associated with thrombotic disorders?

Validating SERPINC1 antibody specificity for mutant variant detection requires a rigorous multi-step approach, especially when studying clinically relevant mutations associated with thrombotic disorders :

• Expression system validation:

  • Generate wild-type and mutant SERPINC1 constructs using site-directed mutagenesis

  • Express in appropriate cell systems (HEK293T cells) with varying expression levels

  • Compare antibody reactivity across variants through western blotting and ELISA

• Epitope mapping assessment:

  • Analyze if mutations fall within or near the antibody's target epitope (amino acids 37-180)

  • For mutations in the epitope region, perform competitive binding assays with synthetic peptides

  • Consider using multiple antibodies targeting different epitopes for complete coverage

• Cross-validation approaches:

  • Compare results with mass spectrometry for unambiguous protein identification

  • Use gene editing (CRISPR/Cas9) to create cell lines with endogenous mutations

  • Perform immunoprecipitation followed by protein sequencing to confirm target identification

• Clinical sample verification:

  • Test antibody performance on samples from patients with known SERPINC1 mutations

  • Compare antibody-based detection with functional activity assays

  • Establish detection thresholds for different mutation types (missense vs. truncating)

• Structural impact analysis:

  • Use structural biology techniques to determine if mutations alter protein conformation

  • Assess if conformational changes affect antibody accessibility to epitopes

  • Correlate structural predictions with experimental antibody binding data

This comprehensive validation strategy ensures that researchers can confidently employ SERPINC1 antibodies for studying clinically relevant mutations, providing reliable data for both research and potential diagnostic applications.