TFPI2 Antibody

What is TFPI2 and why is it significant in oncology research?

TFPI2, also known as placental protein 5 (PP5) or retinal pigment epithelium cell factor 1 (REF1), is a 27 kDa secreted protein with potent tumor suppressor functions. TFPI2 contains three Kunitz domains (residues 36-86, 96-149, and 158-208) separated by two linker regions, with N-terminal acidic and C-terminal basic regions .

This protein has attracted significant research interest because:

• It functions as a protease inhibitor that regulates plasmin-mediated matrix remodeling

• It inhibits trypsin, plasmin, factor VIIa/tissue factor complex, and weakly inhibits factor Xa

• Low expression correlates with enhanced tumor growth and metastasis across multiple cancer types

• It has dual localization (cytoplasmic and nuclear) with distinct functions in each compartment

• It shows promise as a biomarker, particularly in ovarian clear cell carcinoma

Which applications are validated for TFPI2 antibodies?

TFPI2 antibodies have been validated across multiple experimental platforms:

Different antibody clones may perform optimally in specific applications; therefore, selecting an antibody validated for your particular application is essential .

What cell lines and tissue samples can be used as positive controls for TFPI2 antibody validation?

Based on confirmed TFPI2 expression patterns, the following samples serve as reliable positive controls:

Cell Lines:

• Jurkat (human T cell leukemia)

• MCF7 (human breast adenocarcinoma)

• HeLa (human cervical adenocarcinoma)

Tissue Samples:

• Mouse brain, heart, kidney, and spleen lysates

• Rat spleen lysate

• Human kidney tissue sections

• Ovarian clear cell carcinoma (OCCC) tissues

When validating a new TFPI2 antibody, running appropriate positive controls alongside experimental samples is crucial for determining specificity and sensitivity .

What are recommended protocols for Western blotting with TFPI2 antibodies?

Recommended Western Blotting Protocol:

• Sample Preparation:

  • Prepare cell/tissue lysates using standard RIPA buffer with protease inhibitors

  • Load 10-20 μg protein per lane

• Gel Electrophoresis and Transfer:

  • Use 10-12% SDS-PAGE gels for optimal separation

  • Transfer to PVDF or nitrocellulose membranes

• Blocking:

  • Block in 5% non-fat dry milk (NFDM) in TBST for 1 hour at room temperature

• Primary Antibody Incubation:

  • Dilute TFPI2 antibody in 5% NFDM/TBST

  • Recommended dilutions vary by antibody:

    • EPR14442 clone: 1/5000-1/10000

    • Polyclonal antibodies: 1/1000

  • Incubate overnight at 4°C

• Detection:

  • Use HRP-conjugated secondary antibodies at 1/1000 dilution

  • Expected band size: 27 kDa

Multiple TFPI2 isoforms or post-translational modifications may result in additional bands. Validate any unexpected bands using positive and negative controls .

How can I distinguish between nuclear and cytoplasmic TFPI2 in experimental analyses?

TFPI2 exhibits dual localization with distinct functions in different cellular compartments. To specifically study compartment-specific TFPI2:

For Microscopy-based Detection:

• Use immunofluorescence with confocal microscopy

• Co-stain with nuclear markers (DAPI/Hoechst) and cytoplasmic markers

• Analyze co-localization using image analysis software

For Biochemical Fractionation:

• Perform subcellular fractionation to separate nuclear and cytoplasmic fractions

• Verify fraction purity using compartment-specific markers (e.g., lamin for nucleus, GAPDH for cytoplasm)

• Analyze TFPI2 distribution by Western blotting

Functional Validation:

• Nuclear TFPI2 functions as a transcriptional repressor of MMP-2

• Cytoplasmic TFPI2 interacts with actinin-4 and myosin-9

Research has shown that the translocation of TFPI2 into cell nuclei represses transcription of the MMP-2 gene, resulting in reduced invasive ability of breast cancer cells . Meanwhile, cytoplasmic TFPI2 interacts with proteins involved in cell migration and invasion .

What experimental approaches can determine TFPI2 protein-protein interactions?

Co-immunoprecipitation (Co-IP) Protocol:

• Sample Preparation:

  • Prepare cytosolic extracts using non-denaturing lysis buffer

  • Use 500-1000 μg total protein per IP reaction

• Antibody Coupling:

  • Options include:

    • Direct pull-down using agarose-conjugated TFPI2 antibodies

    • Pre-couple unconjugated antibodies to Protein A/G beads

    • For His-tagged TFPI2, use His-Bind resin

• Precipitation:

  • Incubate lysates with antibody-coupled beads overnight at 4°C

  • Wash extensively with buffer containing mild detergent

  • Elute bound proteins and analyze by Western blotting

• Confirmation by Reciprocal Co-IP:

  • Perform reverse Co-IP using antibodies against identified interacting proteins

  • Example: Co-IP using antibodies against actinin-4 or myosin-9 can pull down TFPI2

Mass Spectrometry Identification:

• For novel interaction discovery, analyze Co-IP samples by MS/MS

• Researchers have identified actinin-4 and myosin-9 as TFPI2-interacting proteins with coverage rates of 60.2% and 52.2%, respectively

Verification Techniques:

• Confirming interactions through GST pull-down assays

• Mapping interaction domains using truncated TFPI2 constructs

Research has established that full-length TFPI2 is required for binding to actinin-4, whereas only the N+KD1 regions are sufficient to interact with myosin-9 .

How can I develop a quantitative ELISA for measuring TFPI2 in biological samples?

Sandwich ELISA Development Protocol:

• Antibody Selection:

  • Use two different anti-TFPI2 monoclonal antibodies recognizing distinct epitopes:

    • Capture antibody: Immobilized on plate or magnetic beads

    • Detection antibody: Labeled with enzyme (e.g., alkaline phosphatase)

• Assay Format:

  • One-step immunofluorometric format is effective for TFPI2

  • Alternatively, standard indirect sandwich ELISA can be used

• Calibration Standards:

  • Prepare recombinant TFPI2 protein as calibration standard

  • Example: recombinant TFPI2 from CM of transfected SP2/0 cells

  • Create serial dilutions in sample dilution buffer

• Sample Processing:

  • For cell culture medium: Centrifuge to remove debris

  • For serum/plasma: Dilute appropriately in assay buffer

  • For tissue lysates: Homogenize in non-denaturing buffer

• Assay Validation:

  • Determine lower limit of detection and quantification

  • Assess linearity, precision, and recovery

  • Confirm specificity using TFPI2-depleted samples

This approach has been successfully used on automated immunoassay analyzer systems for clinical sample testing .

What techniques can assess TFPI2's role in the tumor microenvironment, particularly in glioblastoma models?

Experimental Approaches for Tumor Microenvironment Studies:

• Co-culture Systems:

  • Establish co-cultures of glioblastoma stem cells (GSCs) with microglia

  • Modulate TFPI2 expression using shRNA or overexpression

  • Analyze effects on microglia migration, polarization, and function

• Conditioned Media (CM) Experiments:

  • Collect CM from TFPI2-overexpressing or TFPI2-depleted GSCs

  • Treat microglia with CM and assess:

    • Migration (using transwell assays)

    • Immunosuppressive polarization (CD206+ cells by flow cytometry)

    • Marker expression by qRT-PCR

• In Vivo Models:

  • Establish orthotopic glioblastoma models using TFPI2-modified cells

  • Analyze tumor microenvironment components:

    • Microglia: CD45lowCD11b+CX3CR1+ cells

    • Macrophages: CD45highCD11b+CD68+ cells

    • T cells: CD3+, CD4+, CD8+ populations

• Signaling Pathway Analysis:

  • Evaluate TFPI2's effect on STAT6 signaling in microglia

  • Use STAT6 inhibitors (e.g., AS1517499) to block TFPI2-induced effects

  • Measure expression of immunosuppressive markers (CD206, ARG1)

Research has demonstrated that GSC-derived TFPI2 promotes microglia infiltration and immunosuppressive polarization in GBM through CD51-STAT6 signaling pathway activation . This creates an immunosuppressive microenvironment that inhibits T-cell function and promotes tumor growth .

How can I study the mechanism of TFPI2-mediated tumor suppression using antibody-based approaches?

Experimental Strategies:

• ERK1/2 Signaling Analysis:

  • Transfect cells with TFPI2 expression vectors or siRNA

  • Analyze ERK1/2 phosphorylation by Western blotting

  • Examine subcellular localization of p-ERK1/2 by immunofluorescence

  • Correlate with cell proliferation assays

• Functional Domain Mapping:

  • Generate truncated TFPI2 constructs (e.g., N+KD1, full-length)

  • Perform Co-IP to identify domain-specific interactions

  • Assess effects on cell migration and invasion

• Transcriptional Repression Studies:

  • Analyze MMP-2 expression after nuclear translocation of TFPI2

  • Perform chromatin immunoprecipitation (ChIP) to detect TFPI2 at MMP-2 promoter

  • Correlate with invasive ability of cancer cells

• Cytoskeletal Protein Interactions:

  • Study TFPI2 interactions with actinin-4 and myosin-9

  • Evaluate effects on cytoskeletal organization and cell motility

  • Use siRNA-mediated knockdown of interaction partners to assess functional relevance

Research has established that overexpression of TFPI2 decreases phosphorylation of ERK1/2 and inhibits translocation of p-ERK1/2 from cytoplasm to nucleus, ultimately reducing cell proliferation . Additionally, TFPI2 suppresses cancer cell invasion through interactions with cytoskeletal proteins actinin-4 and myosin-9, though these interactions occur independently (TFPI2 does not simultaneously complex with both proteins) .

What are common issues when using TFPI2 antibodies in Western blotting and how can they be resolved?

Common Issues and Solutions:

For optimal results, validate TFPI2 antibodies using known positive control samples such as Jurkat, MCF7, or HeLa cell lysates .

What factors influence TFPI2 detection in immunohistochemistry?

Critical Factors for IHC Optimization:

• Antigen Retrieval:

  • Heat-mediated antigen retrieval with citrate buffer (pH 6) is recommended

  • Insufficient retrieval is a primary cause of weak or absent staining

• Antibody Selection:

  • Different clones may have different optimal conditions

  • Monoclonal antibodies typically provide more consistent results

  • Polyclonal antibodies may offer higher sensitivity but variable specificity

• Tissue Fixation:

  • Overfixation can mask epitopes

  • Standard fixation: 10% neutral buffered formalin for 24-48 hours

• Antibody Dilution:

  • Start with 1/250 dilution for IHC-P

  • Titrate as needed for optimal signal-to-noise ratio

• Detection System:

  • HRP-polymer systems offer superior sensitivity

  • Avoid excessive DAB development which can increase background

• Counterstaining:

  • Hematoxylin counterstain provides good nuclear contrast

  • Adjust counterstaining time to avoid masking specific signals

Researchers have successfully used these approaches for detecting TFPI2 in human kidney tissue sections and in ovarian clear cell carcinoma samples .

How can TFPI2 antibodies be used to evaluate cancer stem cell properties in glioblastoma research?

TFPI2 has been identified as a critical factor in glioblastoma stem cell (GSC) maintenance and tumor immunosuppression. Antibody-based approaches to study this include:

Experimental Approaches:

• GSC Self-Renewal Assessment:

  • Use immunofluorescence to correlate TFPI2 expression with stemness markers

  • Analyze JNK-STAT3 pathway activation in TFPI2-expressing cells

  • Compare tumorsphere formation capacity with TFPI2 expression levels

• Tumor Microenvironment Analysis:

  • Utilize multi-color immunofluorescence to simultaneously detect:

    • TFPI2-expressing GSCs

    • CX3CR1+ microglia

    • CD206+/CD163+/ARG1+ immunosuppressive microglia

    • CD8+/CD4+ T cells

• Signaling Pathway Studies:

  • Evaluate phosphorylation of STAT3 in GSCs expressing TFPI2

  • Assess STAT6 activation in microglia treated with TFPI2

• Therapeutic Targeting Assessment:

  • Use TFPI2 antibodies to monitor protein levels after treatment with:

    • STAT3 inhibitors (to block GSC self-renewal)

    • STAT6 inhibitors (to block microglial immunosuppressive polarization)

    • Immune checkpoint inhibitors (e.g., anti-PD1)

Research has demonstrated that inhibition of the TFPI2-CD51-STAT6 signaling axis activates T cells and synergizes with anti-PD1 therapy in glioblastoma mouse models, suggesting therapeutic potential in targeting this pathway .

How can TFPI2 antibodies aid in developing biomarkers for ovarian clear cell carcinoma?

TFPI2 has emerged as a promising serum biomarker for ovarian clear cell carcinoma (OCCC). Antibody-based approaches include:

Biomarker Development Strategies:

• Sandwich Immunoassay Development:

  • Utilize two different anti-TFPI2 monoclonal antibodies

  • Optimize one-step immune fluorometric assay format

  • Calibrate using recombinant TFPI2 standards

• Tissue Expression Analysis:

  • Perform IHC on formalin-fixed, paraffin-embedded tissues

  • Compare TFPI2 expression across different epithelial ovarian cancer subtypes

  • Correlate expression with clinical outcomes

• Secreted TFPI2 Quantification:

  • Analyze TFPI2 levels in conditioned media (CM) from cancer cell lines

  • Compare TFPI2 concentration in patient serum samples

  • Establish normal reference ranges and diagnostic cutoffs

• Validation in Clinical Cohorts:

  • Test sensitivity and specificity in diverse patient populations

  • Evaluate TFPI2 as a complement to existing biomarkers

  • Assess prognostic value through survival analysis

Researchers have examined TFPI2 expression in 142 epithelial ovarian cancer patients, including 77 OCCC cases, demonstrating its potential as a discriminatory biomarker .

What are the approaches for studying TFPI2's dual roles in coagulation and cancer biology?

TFPI2 functions at the intersection of coagulation and cancer biology, requiring specialized approaches to study these distinct but related roles:

Experimental Strategies:

• Protease Inhibition Assays:

  • Measure TFPI2's inhibitory activity against:

    • Trypsin

    • Plasmin

    • Factor VIIa/tissue factor complex

    • Factor Xa

  • Compare wild-type versus mutant TFPI2 constructs

• Matrix Remodeling Assessment:

  • Analyze plasmin-mediated matrix degradation in the presence/absence of TFPI2

  • Evaluate MMP activity after modulating TFPI2 expression

  • Study ECM component turnover in 3D culture systems

• Integration of Coagulation and Cancer Pathways:

  • Investigate TFPI2 interactions with plasma lipoproteins

  • Assess how coagulation factors influence cancer cell invasion

  • Determine if TFPI2's anti-invasive properties relate to its anti-proteolytic function

• Structure-Function Analysis:

  • Map the functional importance of TFPI2's three Kunitz domains

  • Determine which domains are critical for:

    • Protease inhibition

    • Cell signaling

    • Protein-protein interactions

Research has established that TFPI2's Kunitz domains are crucial for its protease inhibitory functions, while its interactions with cytoskeletal proteins like actinin-4 and myosin-9 appear to mediate its effects on cell migration and invasion .

This comprehensive understanding can guide the development of therapeutic strategies targeting TFPI2 for both thrombotic disorders and cancer.