SERPINA5 Antibody, FITC conjugated

Basic Research Questions

• What is SERPINA5 and what biological functions does it serve in research models?

  SERPINA5, also known as Protein C inhibitor (PCI) or PAI-3, is a heparin-dependent serine protease inhibitor that belongs to the serpin family. It functions primarily in body fluids and secretions, playing crucial roles in regulating proteolytic activities both within and outside blood vessels.

  Functionally, SERPINA5 inhibits serine proteases by irreversibly binding to their active sites. It exhibits dual functions in coagulation pathways - acting as both a procoagulant by inhibiting the anticoagulant activated protein C, and as an anticoagulant by inhibiting blood coagulation factors including prothrombin, factor XI, factor Xa, and plasma kallikrein .

  Recent research has revealed its unexpected role in innate immunity, where it functions as an interferon-stimulated gene (ISG). Studies have demonstrated that SERPINA5 can trigger antiviral immunity by regulating the phosphorylation and nuclear translocation of STAT1, thus activating interferon-related signaling pathways .

• What applications is the SERPINA5 Antibody, FITC conjugated suitable for?

  SERPINA5 Antibody, FITC conjugated has been validated for multiple research applications:

  • Western Blotting (WB): Detects SERPINA5 protein with high specificity in protein lysates

  • Immunohistochemistry (IHC): Allows visualization of SERPINA5 expression in paraffin-embedded tissue sections

  • Immunofluorescence (IF): The FITC conjugation particularly suits direct fluorescence microscopy at dilutions of 1:50-1:200

  • Flow Cytometry: Useful for analyzing SERPINA5 expression in cell populations

  • ELISA: Some products are validated for ELISA applications with recommended dilutions around 1:20000

  Methodologically, researchers should implement a titration series when first using the antibody in any application to determine optimal concentration for their specific experimental conditions.

• How should SERPINA5 Antibody, FITC conjugated be properly stored and handled to maintain its functionality?

  For optimal preservation of FITC-conjugated SERPINA5 antibodies, follow these research-validated protocols:

  • Store in light-protected vials or cover with light-protecting material (e.g., aluminum foil) to prevent photobleaching of the FITC fluorophore

  • Maintain at 4°C for short-term storage (up to 12 months)

  • For extended storage (up to 24 months), dilute with up to 50% glycerol and store at -20°C to -80°C

  • Avoid repeated freeze-thaw cycles as they compromise both enzyme activity and antibody binding capacity

  • For working solutions, prepare fresh dilutions on the day of experiment

  • When conducting immunofluorescence experiments, minimize exposure to light during all handling steps

  Research has demonstrated that proper storage significantly impacts experimental reproducibility, particularly for fluorophore-conjugated antibodies.

• What sample preparations are optimal for detecting SERPINA5 in different tissue and cell types?

  Sample preparation protocols vary by application and tissue source:

  For tissues in IHC:

  • Heat-mediated antigen retrieval in EDTA buffer (pH 8.0) is recommended based on validated protocols

  • Block tissue sections with 10% goat serum to minimize non-specific binding

  • Incubate with primary SERPINA5 antibody (typically 1 μg/ml) overnight at 4°C

  For cells in immunofluorescence:

  • Fix cells with 4% paraformaldehyde followed by permeabilization with 0.1-0.5% Triton X-100

  • For intracellular staining in flow cytometry, cells should be fixed with appropriate fixation buffer and permeabilized before antibody staining

  For Western blot:

  • Sample preparation from tissue requires homogenization in RIPA buffer with protease inhibitors

  • Protein concentration should be determined by BCA or Bradford assay

  • Typically 20-50 μg of total protein is loaded per lane for optimal SERPINA5 detection

• What are the species reactivity profiles for commercially available SERPINA5 antibodies?

  SERPINA5 antibodies show variable species reactivity profiles depending on the specific product:

  • The majority of antibodies have confirmed reactivity to Human SERPINA5

  • Some antibodies demonstrate cross-reactivity with Mouse and Rat samples

  • Broader species reactivity has been reported for certain polyclonal antibodies, with predicted reactivity (based on sequence homology) to Cow (100%), Dog (100%), Guinea Pig (100%), Horse (100%), Rabbit (83%), and Rat (100%)

  When selecting an antibody for non-human research models, it's advisable to verify the specific epitope sequence conservation across species to ensure experimental validity. The immunogen peptide sequence information, when available, provides insight into potential cross-reactivity .

Advanced Research Questions

• How can SERPINA5 expression patterns be accurately quantified in cancer research models?

  Quantification of SERPINA5 expression in cancer models requires multi-method validation:

  For gene expression analysis:

  • qRT-PCR using validated primers targeting conserved regions of SERPINA5 mRNA has proven effective in gastric cancer research, where SERPINA5 shows significant upregulation compared to normal tissues

  • RNA-seq data analysis should incorporate proper normalization with housekeeping genes

  For protein quantification:

  • Western blotting with densitometric analysis calibrated against internal controls

  • ELISA-based quantification using commercially available kits with detection ranges of 0.156-10 ng/mL and sensitivity of 0.039 ng/mL

  • For tissue microarrays, digital image analysis of IHC staining intensity and distribution

  The differential expression of SERPINA5 observed in gastric cancer (higher in tumor vs. normal tissues) contrasts with its reported lower expression in kidney tumors , suggesting tissue-specific regulatory mechanisms that warrant careful methodological consideration in experimental design.

• What experimental approaches can effectively study the role of SERPINA5 in antiviral immunity?

  Research on SERPINA5's role in antiviral immunity can be approached through several methodologies:

  For in vitro models:

  • Gene knockdown using siRNA (siRNA-820 has shown high knockdown efficiency) to assess SERPINA5's impact on viral replication

  • Overexpression systems (e.g., 293T-SerpinA5 stable cell lines) for gain-of-function studies

  • IFN signaling pathway analysis using luciferase reporter systems with IFN-β and ISRE promoters

  For pathway analysis:

  • Co-immunoprecipitation assays to investigate protein-protein interactions, particularly with STAT1

  • Subcellular fractionation and immunoblotting to track STAT1 phosphorylation and nuclear translocation

  • RNA-seq to identify differentially expressed genes in SERPINA5-manipulated cells

  Time-course experiments have shown that SERPINA5 expression increases in response to TLR agonists and viral infections in a time-dependent manner, with this upregulation being IFN-dependent . This suggests that temporal dynamics are critical when designing experiments.

• How should researchers troubleshoot inconsistent results when using SERPINA5 antibodies in different applications?

  When facing inconsistent results with SERPINA5 antibodies, implement this systematic troubleshooting approach:

  For Western blot inconsistencies:

  • Verify antibody specificity using positive controls (e.g., HepG2 cells or liver tissue)

  • Optimize protein extraction methods - SERPINA5 may require specific buffer conditions

  • Test multiple antibody dilutions ranging from 1:500 to 1:5000

  • Compare results using different detection systems (chemiluminescence vs. fluorescence)

  For IHC/IF variability:

  • Compare different antigen retrieval methods (heat-mediated EDTA vs. citrate buffer)

  • Evaluate alternative blocking reagents if background is problematic

  • Test both monoclonal and polyclonal antibodies against different epitopes

  • Consider dual staining approaches to confirm specificity

  Inter-laboratory validation studies have shown that antibody performance can vary significantly based on lot, storage conditions, and detection methodology. Establishing a standardized protocol with appropriate controls is essential for reproducible results.

• What methodological considerations are important when studying SERPINA5's role in tumor cell proliferation?

  When investigating SERPINA5's involvement in tumor cell proliferation:

  Experimental design should include:

  • Multiple cell proliferation assays (MTT/MTS, colony formation, BrdU incorporation) to corroborate findings

  • Cell cycle analysis by flow cytometry to determine specific phase effects

  • Apoptosis assessment using Annexin V-FITC/PI staining to distinguish between antiproliferative and pro-apoptotic effects

  Molecular mechanism investigation:

  • Analyze pathway components downstream of SERPINA5, particularly the Akt/mTOR pathway proteins:

    • Phosphorylated Akt (p-Akt)

    • Phosphorylated mTOR (p-mTOR)

    • Cell cycle regulators (CyclinD1, CDK4)

    • Apoptosis markers (Bax)

  Knockdown experiments have demonstrated that silencing SERPINA5 in gastric cancer cell lines (MKN-28 and BGC-823) reduces proliferation and colony formation capacity . Control experiments should include rescue experiments to confirm specificity of observed effects.

• How can researchers design experiments to investigate SERPINA5's interaction with the JAK/STAT pathway in different disease models?

  To investigate SERPINA5's interaction with the JAK/STAT pathway:

  Protein-protein interaction studies:

  • Co-immunoprecipitation assays to confirm direct interaction between SERPINA5 and STAT1

  • Proximity ligation assays for in situ visualization of molecular interactions

  • FRET/BRET analyses for real-time interaction monitoring in living cells

  Signaling pathway dynamics:

  • Time-course experiments examining STAT1 phosphorylation status following SERPINA5 manipulation

  • Nuclear/cytoplasmic fractionation to track STAT1 translocation

  • Chromatin immunoprecipitation (ChIP) to assess STAT1 binding to target gene promoters

  Pharmacological approach:

  • JAK inhibitors (e.g., ruxolitinib) to determine if SERPINA5's effects are JAK-dependent

  • Phosphatase inhibitors to assess involvement of negative regulators

  Disease model considerations:

  • Implement both viral infection models and tumor models where JAK/STAT signaling is relevant

  • Include analysis of additional pathway components (STAT2, IRF9) that may form complexes

  • Compare effects across different cell types (immune cells vs. epithelial cells)

  Research has demonstrated that SERPINA5 can upregulate STAT1 phosphorylation and promote its nuclear translocation, thereby activating interferon-related signaling pathways . This mechanism appears independent of the cGAS-STING pathway, suggesting multiple modes of action that should be considered in experimental design.

Technical Research Questions

• What are the optimal imaging parameters for visualizing FITC-conjugated SERPINA5 antibodies in fluorescence microscopy?

  For optimal visualization of FITC-conjugated SERPINA5 antibodies:

  Microscopy settings:

  • Excitation wavelength: 495 nm

  • Emission wavelength: 519 nm

  • Use appropriate filter sets (FITC/GFP channels)

  • Image acquisition parameters should be standardized across experimental conditions

  Sample preparation considerations:

  • Mount slides with anti-fade mounting medium containing DAPI for nuclear counterstaining

  • For multi-color imaging, select fluorophores with minimal spectral overlap

  • Prepare negative controls (secondary antibody only) to assess background autofluorescence

  Quantitative analysis protocols:

  • Establish consistent threshold settings for signal quantification

  • Employ z-stack imaging for thick specimens to capture total signal

  • Consider photobleaching correction for time-lapse studies

  In published research, FITC-conjugated SERPINA5 antibodies have been successfully used in immunofluorescent analysis of HepG2 cells at a dilution of 1:100 , serving as a useful reference point for initial protocol development.

• What methods can effectively assess the effect of SERPINA5 knockdown on cellular pathways and functions?

  To comprehensively evaluate SERPINA5 knockdown effects:

  Gene silencing approaches:

  • siRNA transfection: siRNA-820 has demonstrated high knockdown efficiency (>70%)

  • shRNA for stable knockdown in long-term studies

  • CRISPR-Cas9 for complete gene knockout when partial silencing is insufficient

  Validation of knockdown efficiency:

  • RT-qPCR for mRNA quantification

  • Western blotting to confirm protein reduction

  Functional analyses:

  • Cell viability and proliferation assays (MTT, colony formation)

  • Migration and invasion assays if studying cancer models

  • Viral replication assays when investigating antiviral functions

  Pathway investigation:

  • RNA-seq or targeted gene expression profiling to identify affected pathways

  • Phosphoprotein analysis focusing on Akt/mTOR and JAK/STAT pathways

  • Rescue experiments by re-introducing SERPINA5 to confirm specificity

  Previous studies have shown that SERPINA5 knockdown in 293T-SerpinA5 cells increased susceptibility to HSV-1 infection , while in gastric cancer cells, knockdown reduced proliferation , highlighting the importance of cellular context in experimental design.

• How can researchers accurately differentiate between specific SERPINA5 antibody binding and non-specific background in complex tissue samples?

  To optimize signal-to-noise ratio and ensure binding specificity:

  Control samples:

  • Negative controls: tissue known to be negative for SERPINA5 expression

  • Isotype controls: non-specific antibody of same isotype and concentration

  • Absorption controls: pre-incubation of antibody with immunizing peptide

  Staining protocol refinements:

  • Optimize blocking conditions (10% goat serum has been validated)

  • Increase washing duration and frequency between steps

  • Titrate primary antibody concentration to minimize background

  • Consider using biotin-streptavidin amplification systems for weak signals

  Advanced validation techniques:

  • Dual-labeling with antibodies against different epitopes

  • Compare staining patterns between monoclonal and polyclonal antibodies

  • Correlate protein detection with mRNA expression (ISH or qPCR from microdissected regions)

  Quantification methods:

  • Implement digital image analysis with appropriate thresholding

  • Use ratiometric approaches comparing signal to background areas

  • Consider spectral unmixing for autofluorescent tissues

  Validated protocols have demonstrated successful SERPINA5 detection in paraffin-embedded sections of mouse and rat kidney tissue using heat-mediated antigen retrieval in EDTA buffer (pH 8.0) .

• What experimental designs can best elucidate the differential expression of SERPINA5 across normal and pathological states?

  To investigate SERPINA5 expression patterns across health and disease:

  Comparative tissue analysis:

  • Paired tumor/normal tissue samples from the same patient to control for individual variability

  • Tissue microarrays for high-throughput screening across multiple patient samples

  • Longitudinal sampling in progressive disease models

  Multi-omics approach:

  • Integrate transcriptomics, proteomics, and functional data

  • Compare mRNA and protein levels to identify post-transcriptional regulation

  • Correlate with clinical parameters for translational relevance

  Single-cell analysis:

  • scRNA-seq to identify cell type-specific expression patterns

  • Multiplex immunofluorescence to assess heterogeneity within tissues

  • Spatial transcriptomics to maintain tissue architecture context

  Data analysis considerations:

  • Use appropriate statistical methods for paired data

  • Implement machine learning approaches for pattern recognition in complex datasets

  • Generate receiver operating characteristic (ROC) curves to assess diagnostic potential

  Research has revealed contrasting expression patterns: SERPINA5 shows increased expression in gastric cancer but decreased expression in kidney tumors , emphasizing the need for tissue-specific analysis rather than generalizing across cancer types.

• How should researchers design experiments to investigate SERPINA5's role in the regulation of inflammatory responses?

  For studying SERPINA5 in inflammatory contexts:

  In vitro inflammation models:

  • Stimulation with TLR agonists (which have been shown to upregulate SERPINA5 expression)

  • Cytokine treatment time-course experiments

  • Co-culture systems with immune and tissue cells

  Cytokine/chemokine profiling:

  • Multiplex assays to assess inflammatory mediators (IFN-β, MX1, CXCL10, IL-1β, IFN-λ)

  • ELISA validation of key targets

  • mRNA expression analysis using RT-qPCR

  Signaling pathway analysis:

  • Focus on IFN-related pathways, particularly JAK/STAT signaling

  • Assess NF-κB pathway components given their central role in inflammation

  • Examine crosstalk between SERPINA5 and other inflammatory regulators

  Functional read-outs:

  • Neutrophil/macrophage migration assays

  • Phagocytosis assessment

  • Inflammasome activation monitoring

  Research has demonstrated that SERPINA5 can function as an interferon-stimulated gene that triggers antiviral immunity by regulating STAT1 phosphorylation and nuclear translocation . This suggests its potential involvement in broader inflammatory responses that should be systematically investigated.