SERPINB2 Antibody, FITC conjugated

What is SERPINB2 and what cellular functions does it regulate?

SERPINB2 (Serpin Family B Member 2), also known as Plasminogen Activator Inhibitor 2 (PAI-2), is a serine protease inhibitor that primarily inhibits urokinase plasminogen activator (uPA). It functions in multiple biological processes including:

• Inhibition of cell migration and invasion through uPA inhibition

• Regulation of immune responses, particularly Th1/Th2 balance

• Modulation of inflammation processes and macrophage function

• Promotion of cellular senescence through p21 stabilization

• Regulation of hemostasis and coagulation

• Control of extracellular matrix remodeling

At the subcellular level, SERPINB2 can be found in the cytoplasm, focal adhesions, lamellipodia, and on microparticles, often associated with actin structures .

How does a FITC-conjugated SERPINB2 antibody compare with unconjugated versions?

A FITC-conjugated SERPINB2 antibody offers several distinct advantages over unconjugated versions:

FITC conjugation enables direct visualization without secondary antibodies, which is particularly valuable for multi-color flow cytometry and live cell applications .

In which tissue and cell types is SERPINB2 predominantly expressed?

SERPINB2 demonstrates distinctive expression patterns across tissues and cell types:

Expression can be significantly upregulated by inflammatory stimuli, PMA treatment, and DNA damage responses . Most detection methods require cell permeabilization as SERPINB2 is predominantly intracellular .

What are the optimal protocols for SERPINB2 detection using FITC-conjugated antibodies in flow cytometry?

For reliable SERPINB2 detection in flow cytometry applications:

Cell Preparation Protocol:

• Harvest cells (1-5 × 10^6 cells per sample)

• Fix with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.1% saponin buffer (critical for cytoplasmic SERPINB2 access)

• Block with 3% BSA in PBS for 30 minutes

• Incubate with FITC-conjugated SERPINB2 antibody (typically 1-5 μg/mL) for 60 minutes at room temperature

• Wash 3× with 0.1% saponin buffer

• Resuspend in appropriate flow buffer and analyze immediately

Critical Parameters:

• Include appropriate controls: isotype control, unstained cells, and ideally SERPINB2 knockout cells (as shown with K562 cells)

• PMA treatment (300nM overnight) can be used as a positive control to upregulate SERPINB2 expression

• For microparticle-associated SERPINB2, adjust FSC/SSC settings to capture smaller events and consider co-staining with Annexin V

• Optimize compensation if multiplexing with other fluorophores, as FITC has significant spectral overlap with PE

This protocol has been validated in human cell lines including K562, Jurkat, and A431 cells .

How can I validate the specificity of a SERPINB2 antibody in my experimental system?

Comprehensive validation of SERPINB2 antibody specificity should include:

Genetic Validation:

• Compare staining between wild-type and SERPINB2 knockout/knockdown cells

  • K562 knockout cells show complete loss of the 40-47 kDa SERPINB2 band

  • SerpinB2−/− and SerpinB2 R380A mouse models provide additional validation tools

Biochemical Validation:

• Western blot analysis showing a single band at the expected molecular weight (45-47 kDa)

• Peptide competition assay using the immunizing peptide

• Immunoprecipitation followed by mass spectrometry confirmation

Functional Validation:

• Induction experiments: Treatment with known SERPINB2 inducers (PMA, inflammatory stimuli)

• Activation-state specificity: Test if antibody recognizes both free SERPINB2 and SERPINB2-protease complexes

• Cross-reactivity assessment with related serpins, particularly other clade B serpins

Application-Specific Validation:

• For fluorescence applications, compare staining patterns with published subcellular localization (cytoplasmic, focal adhesions, lamellipodia)

• Demonstrate co-localization with known interaction partners (actin, p21)

The most rigorous validation combines multiple approaches, with genetic manipulation being the gold standard .

What experimental approaches can distinguish between intracellular and extracellular SERPINB2 pools?

Differentiating intracellular from extracellular SERPINB2 requires specialized methodologies:

Sequential Staining Protocol:

• Non-permeabilized staining (extracellular SERPINB2)

  • Incubate live cells with FITC-conjugated SERPINB2 antibody (1-2 μg/mL)

  • Wash thoroughly with PBS

  • Image or proceed to permeabilization step

• Permeabilized staining (intracellular SERPINB2)

  • Fix cells with 2% PFA for 10 minutes

  • Permeabilize with 0.1% Triton X-100 for 5 minutes

  • Block with 3% BSA

  • Stain with a spectrally distinct SERPINB2 antibody (e.g., with a red fluorophore)

  • Compare the staining patterns between the two steps

Microparticle Isolation Protocol:

• Collect cell culture supernatant or plasma

• Initial centrifugation at 2,500g for 15 minutes to remove cellular debris

• Ultracentrifugation at 20,000g for 30 minutes to pellet microparticles

• Wash microparticle pellet in PBS

• Analyze microparticle-associated SERPINB2 by flow cytometry or immunofluorescence

• Co-stain with annexin V to confirm microparticle identity

Analytical Considerations:

• SERPINB2 lacks a classical secretory signal but reaches the extracellular environment via microparticle formation

• Microparticle-associated SERPINB2 maintains its ability to inhibit urokinase and bind to phosphatidylserine

• Super-resolution microscopy can help distinguish membrane-associated from cytoplasmic SERPINB2

These approaches have been validated in studies examining SERPINB2's role in cancer metastasis and hemostasis .

What are common technical issues with FITC-conjugated SERPINB2 antibodies and how can they be resolved?

For flow cytometry applications, FITC signal can decrease at lower pH; maintain sample buffer at pH 7.4-8.0 for optimal fluorescence intensity .

How do different fixation and permeabilization methods affect SERPINB2 detection?

The choice of fixation and permeabilization significantly impacts SERPINB2 detection:

Experimental Evidence:

• Studies visualizing SERPINB2 in focal adhesions and lamellipodia successfully used PFA fixation with Triton permeabilization

• Microparticle-associated SERPINB2 detection is better preserved with milder fixation and saponin permeabilization

When studying both intracellular and extracellular SERPINB2 pools, consider a combination approach with initial mild fixation followed by controlled permeabilization.

How can I optimize multi-color immunofluorescence panels that include FITC-conjugated SERPINB2 antibodies?

Designing effective multi-color panels with FITC-conjugated SERPINB2 antibodies requires careful consideration:

Spectral Compatibility Table:

Optimized 4-Color Panel for SERPINB2 Studies:

• DAPI - Nuclear DNA

• FITC - SERPINB2

• Texas Red - Phalloidin (actin structures)

• Cy5 - Target protease (uPA/tPA)

Protocol Considerations:

• Sequential Staining Approach:

  • Apply FITC-SERPINB2 antibody first

  • Wash thoroughly

  • Apply remaining antibodies sequentially

  • This minimizes potential cross-reactivity

• Controls Required:

  • Single-color controls for compensation

  • Fluorescence-minus-one (FMO) controls

  • Isotype controls for each fluorophore

  • Absorption controls if multiple rabbit antibodies are used

• Acquisition Settings:

  • Set PMT voltages using single-stained controls

  • Apply appropriate compensation to correct for spillover

  • Use appropriate filter sets (for FITC: 490/530/20 nm)

• Analysis Strategy:

  • Measure colocalization using Pearson's or Mander's coefficients

  • Consider spectral unmixing for highly overlapping fluorophores

  • Use sequential scanning on confocal microscopes to minimize bleed-through

These approaches have been validated in studies examining SERPINB2 colocalization with actin structures in focal adhesions and lamellipodia .

How can FITC-conjugated SERPINB2 antibodies be used to investigate the role of SERPINB2 in cellular migration?

Migration studies with SERPINB2 can be approached through several sophisticated protocols:

Live Cell Migration Assay:

• Seed cells on Matrigel-coated surfaces in migration chambers

• Create a wound/scratch or set up a chemotactic gradient

• Add cell-permeable nuclear dye for tracking

• Conduct time-lapse imaging over 24-48 hours

• Fix cells at different timepoints

• Stain with FITC-SERPINB2 antibody and phalloidin (actin)

• Image using confocal microscopy

• Analyze SERPINB2 localization in:

  • Leading edge lamellipodia

  • Focal adhesions

  • Trailing edge

Quantitative Analysis Methods:

• Measure length of cellular protrusions with and without functional SERPINB2 (wild-type vs. R380A mutant)

• Calculate migration velocity and directionality

• Perform kymograph analysis to track SERPINB2 dynamics at leading edges

• Quantify colocalization between SERPINB2 and actin in focal adhesions (10-25% overlap reported)

Key Findings from Published Research:

• RPM from SerpinB2−/− and SerpinB2 R380A mice migrated significantly faster than wild-type controls

• SERPINB2 consistently localizes near actin in focal adhesions and lamellipodia during migration

• Recombinant SerpinB2, but not SerpinB2 R380A, reduced the length of cellular protrusions in migrating cells

• SERPINB2's inhibitory effect on migration appears dependent on its ability to inhibit uPA

These methodologies have successfully demonstrated SERPINB2's role in regulating cell migration through modulation of the uPA/plasmin system .

What approaches can resolve contradictory data regarding SERPINB2's role in inflammation and immune responses?

To resolve contradictory findings about SERPINB2's immune functions:

Comprehensive Cell-Type Specific Analysis:

• Isolate primary immune cell populations:

  • Monocytes/Macrophages (M1 vs M2 polarized)

  • Dendritic cells (conventional vs. plasmacytoid)

  • T cell subsets (Th1, Th2, Treg)

  • B cells and NK cells

• For each cell type:

  • Measure baseline SERPINB2 expression using FITC-conjugated antibodies

  • Assess expression changes after various stimuli (LPS, IFN-γ, IL-4)

  • Correlate with functional outcomes (cytokine secretion, phagocytosis)

• Compare results across cell types to identify context-dependent patterns

Temporal Expression Analysis Protocol:

• Stimulate cells with inflammatory triggers

• Collect samples at multiple timepoints (0h, 2h, 6h, 12h, 24h, 48h, 72h)

• Analyze SERPINB2 expression by flow cytometry and western blot

• Correlate with markers of different inflammatory phases

• Determine if SERPINB2 has distinct roles during initiation versus resolution

Signaling Pathway Investigation:

• Combine FITC-SERPINB2 staining with phospho-specific antibodies for:

  • NF-κB pathway components (shown to regulate SERPINB2)

  • HMGB1-mediated signaling (identified as upstream regulator)

  • Glucocorticoid receptor pathway (negative regulator of SERPINB2)

• Use gene set enrichment analysis (GSEA) to identify coordinately regulated pathways

Genetic Model Comparison:

• Compare inflammatory responses in:

  • SerpinB2−/− (conventional knockout)

  • SerpinB2 R380A (active site mutant)

  • Wild-type controls

• This approach successfully demonstrated that SerpinB2−/− mice generate ~2.5-fold more OVA-specific IFN-γ-secreting T cells and ~6-fold more IgG2c than controls

These systematic approaches can help reconcile contradictory findings by identifying specific contexts where SERPINB2 promotes versus inhibits inflammation .

How can SERPINB2 antibodies be utilized to study its role in senescence and p21 stabilization?

To investigate SERPINB2's emerging role in senescence:

Comprehensive Senescence Analysis Protocol:

• Establish senescence models:

  • Replicative senescence: Extended cell passaging

  • Stress-induced senescence: Low-dose radiation or chemotherapeutics

  • Oncogene-induced senescence: HRAS-V12 expression

• Confirm senescence phenotype:

  • SA-β-galactosidase staining

  • Cell cycle analysis (G1 arrest)

  • SASP marker expression (IL-6, IL-8)

• SERPINB2 expression analysis:

  • Quantify by flow cytometry with FITC-SERPINB2 antibody

  • Perform Western blot for total protein levels

  • Determine subcellular localization by confocal microscopy

SERPINB2-p21 Interaction Studies:

• Colocalization analysis:

  • Co-stain cells with FITC-SERPINB2 and p21 antibodies

  • Calculate Pearson's correlation coefficient

  • Analyze nuclear vs. cytoplasmic distribution

• Molecular interaction assay:

  • Immunoprecipitate with SERPINB2 antibody

  • Western blot for p21 co-precipitation

  • Perform proximity ligation assay (PLA) for in situ detection

• p21 stability experiments:

  • Treat cells with cycloheximide to block protein synthesis

  • Monitor p21 degradation kinetics in SERPINB2+/+ vs. SERPINB2-/- cells

  • Compare proteasome-dependent and independent degradation pathways

Manipulation Experiments:

• SERPINB2 overexpression:

  • Transfect proliferating cells with SERPINB2 expression vectors

  • Monitor senescence induction (confirmed to occur)

  • Measure p21 protein levels and stability

• SERPINB2 knockdown:

  • siRNA or CRISPR-based SERPINB2 depletion in senescent cells

  • Assess impact on p21 levels and senescence maintenance

  • Determine if senescence reversal occurs

Published research has demonstrated that SERPINB2 directly binds and stabilizes p21 in senescent cells through a proteasome-independent mechanism, and elevated SERPINB2 alone can induce senescence in proliferating cells .

What methodological approaches can distinguish between protease-dependent and protease-independent functions of SERPINB2?

To differentiate SERPINB2's diverse mechanisms of action:

Active Site Mutant Comparison:

• Compare SERPINB2 wild-type with SERPINB2 R380A (active site mutant)

• Both versions can be detected with standard SERPINB2 antibodies

• Assess differential effects on:

  • Cell migration (confirmed protease-dependent)

  • p21 stabilization (protease-independent)

  • Immune modulation (mixed mechanisms)

Complex-Specific Detection:

• Develop or obtain antibodies specific to:

  • Free SERPINB2

  • SERPINB2-uPA complexes

  • SERPINB2-tPA complexes

• Compare distribution patterns across different biological contexts

• Correlate complex formation with functional outcomes

Functional Assays with Comparative Analysis:

Protease Activity Mapping:

• Combine FITC-SERPINB2 staining with fluorogenic uPA/tPA substrates

• Map areas of active proteolysis versus SERPINB2 localization

• Compare inhibition patterns between wild-type and R380A SERPINB2

This systematic approach has successfully distinguished SERPINB2's protease-dependent role in cell migration from its protease-independent functions in senescence .

How can researchers accurately analyze SERPINB2 expression on microparticles?

Microparticle-associated SERPINB2 requires specialized detection methods:

Microparticle Isolation Protocol:

• Collect culture supernatant or plasma (citrate anticoagulant preferred)

• Initial centrifugation: 2,500g for 15 minutes to remove cells and debris

• Second centrifugation: 20,000g for 30 minutes to pellet microparticles

• Wash microparticle pellet with filtered PBS

• Resuspend in buffer appropriate for downstream applications

Flow Cytometry Analysis:

• Adjust flow cytometer settings to detect small particles:

  • Reduce threshold on FSC/SSC

  • Use size-calibrated beads (0.5-1.0 μm) as reference

• Staining protocol:

  • For surface SERPINB2: Incubate microparticles with FITC-SERPINB2 antibody

  • For total SERPINB2: Add mild permeabilization step

  • Include Annexin V (different fluorophore) to confirm phosphatidylserine exposure

• Gating strategy:

  • Gate on size-appropriate events (0.5-1 μm)

  • Confirm microparticle identity with Annexin V positivity

  • Analyze SERPINB2 expression within this population

Advanced Microscopy Approaches:

• Microparticle adherence:

  • Spin microparticles onto poly-L-lysine coated slides

  • Fix with 2% PFA (to preserve structure)

  • Stain with FITC-SERPINB2 antibody

• Super-resolution imaging:

  • Use STORM or STED microscopy for detailed visualization

  • Co-stain with membrane markers to confirm surface localization

Functional Validation:

• Urokinase inhibition assay:

  • Isolate microparticles from cells expressing wild-type or R380A SERPINB2

  • Measure uPA activity using chromogenic or fluorogenic substrates

  • Confirm that microparticle-associated wild-type SERPINB2 inhibits uPA

Research has demonstrated that SERPINB2 is present on microparticles sized 0.5-1 μm, where it maintains its ability to inhibit urokinase and likely binds to phosphatidylserine through annexin interactions .

What experimental design is optimal for studying SERPINB2's role in cancer progression and metastasis?

For comprehensive investigation of SERPINB2 in cancer:

In Vitro Experimental Design:

• Cell model selection:

  • Compare SERPINB2-high vs. SERPINB2-low cancer cell lines

  • Generate stable SERPINB2 overexpression and knockout variants

  • Include both wild-type and R380A SERPINB2 to distinguish mechanisms

• Migration/invasion assays:

  • Transwell invasion through Matrigel

  • 3D spheroid invasion assays

  • Real-time cell analysis with xCELLigence system

• Cell-ECM interaction studies:

  • Collagen gel contraction assays (disrupted by SERPINB2 deficiency)

  • Adhesion assays to various ECM components

  • Degradation assays using fluorescently labeled ECM

In Vivo Metastasis Models:

• Orthotopic tumor models:

  • Pancreatic cancer cells co-injected with wild-type or SERPINB2-/- fibroblasts

  • Track primary tumor growth and local invasion

  • Analyze ECM organization and remodeling at tumor-stroma interface

• Experimental metastasis:

  • Tail vein injection of SERPINB2-manipulated cancer cells

  • Quantify lung colony formation

  • B16 melanoma model shows reduced metastasis with SERPINB2 expression

• Circulating tumor cell (CTC) analysis:

  • Isolate CTCs from peripheral blood

  • Analyze SERPINB2 expression by flow cytometry

  • Compare SERPINB2 levels between primary tumor, CTCs, and metastases

Stromal Component Analysis:

• Dual immunofluorescence:

  • FITC-SERPINB2 antibody

  • Cell-type markers (αSMA for CAFs, CD68 for TAMs)

  • ECM components (collagens, fibronectin)

• Stromal compartment manipulation:

  • Co-injection experiments with modified stromal cells

  • Analysis of stromal SERPINB2 on tumor progression

  • Gene expression profiling of isolated stromal components

Clinical Correlation Studies:

• Tissue microarray analysis:

  • SERPINB2 immunostaining in primary tumors vs. metastases

  • Correlation with patient survival (SERPINB2 associated with better outcomes)

  • uPA expression analysis (inversely correlated with survival)

• Circulating biomarker studies:

  • Microparticle-associated SERPINB2 in patient plasma

  • Correlation with disease stage and progression

Research has established that SERPINB2 expression, particularly in the stromal compartment, is associated with reduced metastasis and prolonged survival in pancreatic ductal adenocarcinoma through regulation of stromal remodeling and local invasion .

How should researchers interpret discrepancies in SERPINB2 antibody detection between different experimental methods?

When faced with methodological discrepancies:

Systematic Troubleshooting Approach:

Epitope Mapping Considerations:

• Some antibodies target the reactive center loop (affected by protease binding)

• Others target the CD interhelical loop region (less affected by conformational changes)

• Antibodies recognizing amino acids 293-389 or internal regions show good reactivity

Biological Variables Affecting Detection:

• SERPINB2 exists in multiple forms:

  • Free SERPINB2 (45-47 kDa)

  • SERPINB2-protease complexes (higher MW)

  • Potential glycosylation variants

  • Microparticle-associated vs. cytoplasmic forms

• Expression level variability:

  • Highly inducible in response to stimuli

  • Can increase from barely detectable to 1% of total protein

  • PMA treatment (300nM overnight) can be used as a positive control