SERPINA5 Antibody

What is the optimal fixation protocol for immunohistochemistry using SERPINA5 antibodies?

For optimal immunohistochemical staining of SERPINA5, heat-mediated antigen retrieval in EDTA buffer (pH 8.0) is recommended. Based on validated protocols:

• Fix tissue sections using 10% neutral buffered formalin for 24-48 hours

• Embed in paraffin and section at 4-6 μm thickness

• Deparaffinize and rehydrate sections through xylene and graded alcohols

• Perform heat-mediated antigen retrieval in EDTA buffer (pH 8.0) for 20 minutes

• Block with 10% goat serum for 30 minutes at room temperature

• Incubate with primary SERPINA5 antibody at 1 μg/ml concentration overnight at 4°C

• Apply biotinylated secondary antibody for 30 minutes at 37°C

• Develop using Strepavidin-Biotin-Complex with DAB as the chromogen

This protocol has been validated on multiple tissue types including mouse and rat kidney tissues, showing consistent and specific staining patterns.

Which cellular compartments typically show SERPINA5 expression?

SERPINA5 exhibits an extensive tissue distribution pattern with both intracellular and extracellular localization:

• Secreted protein: Found in various body fluids including blood plasma, seminal plasma, and cervicovaginal fluid

• Tissue expression: Primarily synthesized in the liver, but also produced in kidneys and steroid-responsive organs

• Subcellular localization: While predominantly secreted, SERPINA5 can undergo nuclear translocation via its nuclear localization signal in the H-helix region

Flow cytometry studies of HepG2 human hepatocellular carcinoma cells demonstrate that SERPINA5 can be detected intracellularly after proper fixation and permeabilization . When investigating SERPINA5 localization, it's important to use both membrane and cytoplasmic/nuclear markers to fully characterize its distribution.

What are the recommended Western blot conditions for SERPINA5 detection?

For optimal Western blot detection of SERPINA5:

Sample preparation and electrophoresis:

• Load 30 μg of protein lysate per lane under reducing conditions

• Use 5-20% gradient SDS-PAGE gels

• Run at 70V (stacking gel)/90V (resolving gel) for 2-3 hours

Transfer and immunoblotting:

• Transfer proteins to nitrocellulose membrane at 150 mA for 50-90 minutes

• Block with 5% non-fat milk in TBS for 1.5 hours at room temperature

• Incubate with anti-SERPINA5 antibody at 0.5 μg/mL overnight at 4°C

• Wash with TBS-0.1% Tween (3 × 5 minutes)

• Probe with goat anti-rabbit IgG-HRP secondary antibody (1:5000 dilution) for 1.5 hours at room temperature

• Develop using Enhanced Chemiluminescent detection system

Expected band size for SERPINA5 is approximately 46 kDa. The protocol has been validated on various sample types including rat liver tissue lysates and human SKOV3 whole cell lysates.

How does SERPINA5 expression differ between normal and cancer tissues?

SERPINA5 expression shows notable tissue-specific variation in cancer contexts:

In gastric cancer, SERPINA5 knockdown experiments demonstrated reduced cell proliferation ability and decreased colony formation, suggesting an oncogenic role . Conversely, in hepatocellular carcinoma, SERPINA5 reduces metastatic potential by inhibiting cell migration through interaction with fibronectin .

These contradictory findings highlight the context-dependent functions of SERPINA5 across different cancer types, necessitating careful validation in each experimental system.

What controls should be included when using SERPINA5 antibodies for research?

When conducting experiments with SERPINA5 antibodies, include these essential controls:

For Western blot:

• Positive control: Recombinant human SERPINA5 protein

• Tissue/cell positive controls: HepG2 cells or liver tissue lysates (high endogenous expression)

• Knockdown control: Lysates from cells treated with validated SERPINA5 siRNA (siRNA-820 has demonstrated high knockdown efficiency)

• Loading control: β-actin or GAPDH to normalize protein loading

For immunohistochemistry:

• Positive tissue control: Kidney tissues show reliable SERPINA5 staining

• Negative control: Omission of primary antibody

• Isotype control: Matched isotype antibody at same concentration as primary

• Absorption control: Pre-incubation of antibody with recombinant SERPINA5

For flow cytometry:

• Unstained cells

• Isotype control (e.g., MAB003) at matched concentration

• Fixation-only control to assess autofluorescence

• Secondary antibody-only control

How can antibody-based techniques be used to study SERPINA5's role in JAK/STAT signaling?

SERPINA5 has been shown to regulate the JAK/STAT pathway, particularly in antiviral immunity. Methods to investigate this relationship include:

• Co-immunoprecipitation studies:

  • Use anti-SERPINA5 antibodies to pull down protein complexes

  • Probe for STAT1 in immunoprecipitates to confirm direct interaction

  • Validate specificity by confirming SERPINA5 does not interact with STAT2 or IRF9

• Phosphorylation assessment:

  • Western blot analysis using phospho-specific antibodies to examine STAT1 phosphorylation status following SERPINA5 modulation

  • Compare nuclear and cytoplasmic fractions to assess compartment-specific effects

• Immunofluorescence colocalization:

  • Perform dual immunofluorescence staining for SERPINA5 and STAT1

  • Track nuclear translocation of STAT1 following IFN-β stimulation with and without SERPINA5 overexpression

• Promoter activity assays:

  • Combine with luciferase reporter systems containing IFN-β or ISRE promoters

  • Quantify effects of SERPINA5 manipulation on promoter activation

Research has demonstrated that SERPINA5 upregulates phosphorylation of STAT1 and promotes its nuclear translocation, thereby activating transcription of IFN-related signaling pathways and enhancing antiviral activity .

How do I optimize immunoprecipitation protocols to study SERPINA5 interactions with extracellular matrix proteins?

SERPINA5 has been shown to interact with fibronectin, impacting cell migration. Optimizing immunoprecipitation for these interactions requires:

Protocol optimization:

• Select appropriate lysis buffer:

  • For secreted proteins: Collect conditioned medium and spike with recombinant SERPINA5 as positive control

  • For matrix proteins: Use buffer containing 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, plus protease inhibitors

• Pre-clearing step:

  • Incubate lysates with protein A/G beads for 1 hour at 4°C to reduce non-specific binding

  • For fibronectin interaction studies, pre-clear with non-immune IgG of the same species as the IP antibody

• Antibody selection considerations:

  • Use antibodies targeting different epitopes of SERPINA5 (N-terminal vs. C-terminal)

  • Consider the region spanned by amino acids 157-367, which has proven effective in multiple applications

• Elution conditions:

  • Acidic elution (pH 2.5-3.0) followed by immediate neutralization

  • Alternatively, competitive elution with specific peptides

  • Gentle elution preferred for maintaining protein-protein interactions

• Controls:

  • Input (5-10% of starting material)

  • IgG control (same amount as IP antibody)

  • Reverse IP (using anti-fibronectin antibody to pull down SERPINA5)

For fibronectin-SERPINA5 interaction specifically, add 100 μg/ml fibronectin in serum-free media when studying effects on cell migration .

What experimental approaches can resolve contradictory findings about SERPINA5's role in different cancer types?

To address the tissue-specific and seemingly contradictory roles of SERPINA5 in cancer:

• Comprehensive tissue analysis across cancer types:

  • Employ tissue microarrays with paired normal/tumor samples

  • Quantify SERPINA5 protein using standardized IHC scoring

  • Correlate with clinical outcomes across cancer types

• Mechanistic pathway assessment:

  • In gastric cancer: Focus on PI3K/AKT/mTOR pathway interactions

  • In hepatocellular carcinoma: Examine fibronectin-integrin signaling

  • Assess CBL inhibition in both contexts to determine if this mechanism is conserved

• Phenotypic assays to distinguish context-dependent functions:

  Cancer Type	Key Mechanism	Recommended Assay	Readout
  Gastric	PI3K/AKT regulation	MTT and colony formation	Cell proliferation
  Liver	Fibronectin interaction	Transwell migration	Cell migration
  Multiple	Inflammation modulation	Cytokine profiling	Tumor microenvironment

• Domain-specific function analysis:

  • Generate SERPINA5 mutants targeting specific functional domains

  • Assess behavior in different cellular contexts

  • Evaluate nuclear vs. cytoplasmic functions separately

• Comprehensive RNA-seq analysis:

  • Compare transcriptional changes induced by SERPINA5 modulation across cancer types

  • Identify cancer-specific gene signatures and pathway enrichment

  • Validate key differentially expressed genes

The seemingly contradictory findings may reflect genuine biological differences in SERPINA5 function dependent on tissue context, expression level, and interacting partners.

How can SERPINA5 antibodies be applied to study its role in antiviral immunity and innate immune signaling?

SERPINA5 has recently been identified as an interferon-stimulated gene (ISG) with antiviral properties. To investigate this function:

• Monitoring SERPINA5 induction during viral infection:

  • Stimulate cells with TLR agonists (LPS, PolyI:C, R848), interferon α, or viral infection

  • Track time-dependent changes in SERPINA5 expression using Western blot and qPCR

  • Compare responses in wild-type vs. IFNAR-deficient cells to confirm IFN-dependence

• Viral infection models:

  • Transfect cells with SERPINA5-expressing plasmids prior to viral infection

  • Measure viral replication using qPCR for viral genes (e.g., HSV-1 UL27)

  • Quantify infectious virus using TCID50 assays

  • Compare results across multiple cell types (HeLa, A549, 293T)

• Pathway analysis approaches:

  • Perform RNA-seq on SERPINA5-treated vs. control cells during infection

  • Focus on DEGs in IFN signaling pathway, TNF signaling, and TLR signaling

  • Validate key findings using reporter assays (IFN-β promoter and ISRE promoter)

• STAT1 interaction studies:

  • Use immunofluorescence to track STAT1 nuclear translocation following SERPINA5 treatment

  • Perform co-IP to confirm direct SERPINA5-STAT1 interaction

  • Examine phosphorylation status in nuclear vs. cytoplasmic fractions

Experimental data has shown that SERPINA5 enhances the phosphorylation and nuclear translocation of STAT1, thereby activating IFN-related signaling pathways that ultimately inhibit viral infections. This represents a previously unrecognized function of SERPINA5 as an antiviral factor .

What methodological approaches can differentiate between SERPINA5's protease inhibitory functions and its signaling roles?

SERPINA5 has dual functionality as a protease inhibitor and a signaling molecule. To distinguish between these roles:

• Domain-specific mutant analysis:

  • Generate reactive center loop (RCL) mutants that lack protease inhibitory activity

  • Create H-helix mutants that affect nuclear localization

  • Test each mutant in both protease inhibition assays and signaling pathway activation

• Differential inhibitor approach:

  • Apply selective protease inhibitors alongside SERPINA5 manipulation

  • Determine if phenotypic effects of SERPINA5 persist when its target proteases are independently inhibited

  • Compare with non-inhibitory SERPINA5 mutants

• In vitro vs. cellular assay comparison:

  • Perform in vitro protease inhibition assays with purified components

  • Compare results with cellular assays measuring signaling pathway activation

  • Analyze concentration-dependency differences between the two functions

• Temporal analysis of SERPINA5 activities:

  • Monitor the kinetics of protease inhibition vs. signaling events

  • Determine if one function precedes and potentially causes the other

  • Use time-course experiments with selective inhibitors of downstream pathways

• Binding partner identification:

  • Perform comprehensive co-IP followed by mass spectrometry

  • Categorize binding partners as protease targets vs. signaling mediators

  • Validate key interactions with reciprocal co-IP and functional assays