TFPI2 Antibody, HRP conjugated

What is TFPI2 and what are its biological functions?

TFPI2 (Tissue Factor Pathway Inhibitor 2) is a Kunitz-type serine proteinase inhibitor that is associated with the extracellular matrix. It functions as a potent tumor suppressor gene that inhibits a variety of serine proteases including factor VIIa/tissue factor, factor Xa, plasmin, trypsin, chymotrypsin, and plasma kallikrein . Low expression of TFPI-2 results in enhanced growth and metastasis of various human tumors .

TFPI2 has multiple biological functions:

• Tumor suppression: TFPI2 suppresses cell proliferation through regulation of ERK signaling pathway .

• Anti-invasion properties: It interacts with proteins like myosin-9 and actinin-4 to inhibit cancer cell invasion .

• Immune modulation: TFPI2 protects against gram-negative bacterial infection and plays a role in host defense .

• Matrix protection: It prevents the proteolytic degradation of the extracellular matrix .

What are the key specifications of TFPI2 Antibody, HRP conjugated?

TFPI2 Antibody, HRP conjugated typically has the following specifications:

• Immunogen: Recombinant Human Tissue factor pathway inhibitor 2 protein (amino acids 23-235) .

• Host species: Available as rabbit polyclonal or mouse monoclonal antibodies.

• Reactivity: Primarily human-specific, though some variants may cross-react with other species .

• Applications: Primarily used in ELISA, but some variants are suitable for Western blot (WB) and immunohistochemistry (IHC) .

• Storage buffer: Typically contains preservatives like 0.03% Proclin 300 and constituents like 50% Glycerol in PBS, pH 7.4 .

• Storage conditions: Recommended storage at -20°C or -80°C, avoiding repeated freeze-thaw cycles .

• Conjugation: Horseradish peroxidase (HRP) for direct detection without secondary antibodies .

How should I optimize TFPI2 Antibody, HRP conjugated for ELISA applications?

For optimal results in ELISA applications:

• Antibody titration: Begin with a dilution series (1:500, 1:1000, 1:2000, 1:5000) to determine optimal concentration that provides adequate signal with minimal background .

• Blocking optimization: Use 1-5% BSA or 3-5% non-fat dry milk in PBS or TBS to reduce non-specific binding.

• Incubation conditions: For primary incubation, maintain a consistent temperature (room temperature or 4°C) and time (1-2 hours at room temperature or overnight at 4°C).

• Substrate selection: Choose the appropriate HRP substrate (TMB, ABTS, or OPD) based on your detection system and sensitivity requirements.

• Validation controls:

  • Include a standard curve using recombinant TFPI2 protein

  • Use positive control samples known to express TFPI2

  • Include negative controls (no primary antibody, no sample)

  • Consider using tissue samples with known differential TFPI2 expression, similar to the hepatocarcinoma vs. normal hepatic tissues comparison showing scores of 22.54±1.22 vs. 46.60±1.80 respectively .

What is the difference between polyclonal and monoclonal TFPI2 antibodies?

Polyclonal TFPI2 antibodies:

• Recognize multiple epitopes on the TFPI2 antigen

• Often raised in rabbits against recombinant human TFPI2 (e.g., AA 23-235)

• May provide higher sensitivity due to binding of multiple epitopes

• Better for detecting denatured proteins in applications like Western blot

• Useful for initial characterization of TFPI2 expression patterns

Monoclonal TFPI2 antibodies:

• Recognize a single epitope on the TFPI2 antigen

• Generated from a single B-cell clone (e.g., OTI1G10 clone)

• Provide consistent lot-to-lot reproducibility

• Higher specificity for a particular domain or region of TFPI2

• More suitable for quantitative assays requiring reproducible results

• Used in applications requiring high specificity like distinguishing between closely related protein domains

The choice between monoclonal and polyclonal antibodies depends on your experimental needs. For screening or detecting TFPI2 in varied contexts, polyclonal antibodies may be preferable. For specific epitope targeting or quantitative analysis, monoclonal antibodies usually offer superior consistency.

How can TFPI2 Antibody, HRP conjugated be used to study protein-protein interactions of TFPI2?

TFPI2 has been shown to interact with several proteins including myosin-9 and actinin-4, which play important roles in cell migration and invasion . To study these interactions:

• Co-immunoprecipitation (Co-IP) followed by immunoblotting:

  • Use TFPI2 antibody to pull down TFPI2 complexes

  • Analyze precipitates by Western blot probing for suspected interaction partners

  • Perform reciprocal Co-IP using antibodies against potential binding partners

• Domain mapping approach:

  • Research has shown that the full-length TFPI-2 associates with both myosin-9 and actinin-4, but truncated forms show differential binding :

    • Full-length TFPI-2 is required for interaction with actinin-4

    • The N+KD1 region of TFPI-2 (amino acids 1-95) is sufficient for interaction with myosin-9

  • Use domain-specific antibodies to determine which regions are critical for specific interactions

• Proximity ligation assay (PLA):

  • Utilize TFPI2 antibody alongside antibodies against potential interaction partners

  • This method allows visualization of protein-protein interactions in situ

• Immunofluorescence co-localization:

  • Use fluorescently labeled antibodies to visualize co-localization of TFPI2 with candidate proteins

  • Follow with confocal microscopy and co-localization analysis

Remember that full-length TFPI-2 does not simultaneously complex with myosin-9 and actinin-4, as indicated by Co-IP experiments , suggesting these interactions may be mutually exclusive or context-dependent.

How can researchers use TFPI2 Antibody to investigate its tumor suppressor function?

TFPI2 has been recognized as a potent tumor suppressor gene, and its expression is reduced in various cancers. To investigate this function:

• Expression analysis in tumor vs. normal tissues:

  • Use immunohistochemistry (IHC) with TFPI2 antibody to compare expression levels

  • In hepatocellular carcinoma studies, TFPI-2 immunostaining scores were significantly lower in tumor tissues (22.54±1.22) compared to normal hepatic tissues (46.60±1.80)

  • Analyze correlation between TFPI2 expression and clinical parameters, as demonstrated in this breast cancer data table:

  Parameters	Number of patients	TFPI2 mRNA expression	P value
  Tumor size: T1-2	26	18 (low) / 8 (high)	0.005*
  Tumor size: T3-4	24	7 (low) / 17 (high)	-
  Metastasis: M0	32	20 (low) / 12 (high)	0.018*
  Metastasis: M1	18	5 (low) / 13 (high)	-
  Pathological stage: I-II	29	19 (low) / 10 (high)	0.010*
  Pathological stage: III-IV	21	6 (low) / 15 (high)	-

• Functional studies:

  • Establish stable cell lines overexpressing TFPI2 in cancer cell models

  • Use TFPI2 antibody to confirm protein expression by Western blot

  • Measure effects on:

    • Cell proliferation using MTT assay (proliferation was inhibited in HepG2-TFPI-2 cells)

    • Cell invasion using Transwell invasion assays

    • ERK1/2 phosphorylation status (TFPI-2 overexpression decreased phosphorylation of ERK1/2)

• Pathway analysis:

  • Examine the effect of TFPI2 on ERK signaling cascade using antibodies against p-ERK1/2

  • Investigate TFPI2's interaction with myosin-9 and actinin-4, which contributes to its suppressive effect on cell invasion

What methodological considerations should be addressed when studying TFPI2 expression in different tissue types?

When studying TFPI2 expression across different tissues, researchers should consider:

• Tissue-specific expression patterns:

  • TFPI-2 shows differential basal expression across tissues

  • During endotoxin-induced inflammation, TFPI-2 expression increases dramatically in specific tissues:

    • 10-fold increase in spleen within 1 hour post-LPS injection

    • Peak expression in brain and lungs at 4 hours post-injection

    • Increased expression also observed in kidney, large and small intestine, and liver

• Sample preparation optimization:

  • For tissue sections: Proper fixation is crucial (typically 10% neutral buffered formalin)

  • Antigen retrieval methods may need tissue-specific optimization (citrate buffer pH 6.0 or EDTA buffer pH 9.0)

  • For cell/tissue lysates: Different extraction buffers may be required for different tissues

• Controls:

  • Include both positive and negative tissue controls

  • For cancer studies, always include paired normal adjacent tissues

  • Consider using TFPI2 knockout models as negative controls

• Detection methods:

  • Combine antibody-based detection with mRNA analysis (RT-PCR, in situ hybridization)

  • In situ hybridization has successfully detected TFPI-2 mRNA in hepatic tissues when paired with immunohistochemistry

• Post-translational modifications:

  • Be aware that TFPI2 can be proteolytically processed, as demonstrated by detection of TFPI-2 fragments in COPD patient sputum samples

  • Consider using antibodies against different domains to detect various forms of the protein

How can TFPI2 Antibody, HRP conjugated be used in multiplex assays with other biomarkers?

For multiplex analysis incorporating TFPI2 with other biomarkers:

• Sequential immunostaining approach:

  • Use TFPI2 antibody, HRP conjugated as the first detection antibody

  • Develop with a chromogenic substrate specific for HRP (e.g., DAB)

  • Perform heat-mediated stripping of the antibody

  • Proceed with subsequent antibodies using different detection systems

  • This approach works well for tissue sections in studies examining multiple markers

• Multiplex ELISA optimization:

  • Use TFPI2 antibody, HRP conjugated alongside other directly labeled antibodies

  • Ensure no cross-reactivity between antibodies (test each antibody individually)

  • Optimize blocking conditions to minimize background across all antibodies

  • Validate antibody combinations using known positive and negative samples

• Co-localization studies:

  • When studying TFPI2's relationship with binding partners like myosin-9 and actinin-4:

    • Use TFPI2 antibody, HRP conjugated with a chromogenic substrate

    • Use fluorescently labeled antibodies for other proteins

    • Perform digital overlay analysis

• Biomarker panel development:

  • When incorporating TFPI2 into cancer biomarker panels:

    • Include markers for related pathways (e.g., ERK signaling components)

    • Include tissue-specific control markers

    • Normalize expression data to appropriate housekeeping proteins

What are the challenges in detecting TFPI2 in clinical samples and how can they be overcome?

Clinical sample analysis presents several challenges for TFPI2 detection:

• Low expression levels:

  • TFPI2 is often downregulated in cancers, making detection difficult

  • Solution: Use signal amplification methods (e.g., tyramide signal amplification) to enhance sensitivity of HRP-conjugated antibodies

  • Consider using more sensitive detection substrates for HRP (e.g., SuperSignal West Femto)

• Sample heterogeneity:

  • Clinical samples often contain mixed cell populations

  • Solution: Combine laser capture microdissection with sensitive detection methods

  • Use dual staining approaches to correlate TFPI2 with cell-type specific markers

• Pre-analytical variables:

  • Time to fixation, fixative type, and processing can affect TFPI2 detection

  • Solution: Standardize pre-analytical protocols and document deviations

  • Validate the antibody with samples processed under different conditions

• Proteolytic processing:

  • TFPI2 can be proteolytically processed in vivo, as demonstrated in COPD patients

  • Solution: Use antibodies targeting different domains of TFPI2

  • Consider Western blot analysis to identify proteolytic fragments

• Quantification challenges:

  • Semi-quantitative methods like IHC scoring have limitations

  • Solution: Implement digital image analysis for more objective quantification

  • Use scoring systems like the one used for hepatocarcinoma tissues (mean score 22.54±1.22) vs. normal hepatic tissues (mean score 46.60±1.80)

How can researchers validate the specificity of TFPI2 Antibody, HRP conjugated?

To ensure antibody specificity:

• Positive and negative controls:

  • Test antibody in tissues/cells known to express high levels of TFPI2 (e.g., normal hepatic tissues)

  • Include negative controls such as tissues/cells with low TFPI2 expression (e.g., hepatocarcinoma tissues)

  • If available, use TFPI2 knockout models as definitive negative controls

• Blocking peptide competition:

  • Pre-incubate the antibody with excess recombinant TFPI2 protein (immunogen)

  • Compare staining patterns with and without blocking peptide

  • Specific staining should be significantly reduced with blocking peptide

• Multiple detection methods:

  • Complement antibody detection with mRNA analysis (RT-PCR or in situ hybridization)

  • Compare protein expression patterns with mRNA expression

  • As demonstrated in hepatocarcinoma research, in situ hybridization and immunohistochemistry showed concordant results

• Validation across applications:

  • Perform Western blot to confirm antibody specificity by molecular weight

  • Verify immunoprecipitation capability with known TFPI2 interaction partners

  • Test antibody in different applications (ELISA, IHC, etc.)

What is the role of TFPI2 in inflammation and how can researchers study this using TFPI2 antibodies?

TFPI2 plays a significant role in inflammation and host defense:

• Expression analysis during inflammation:

  • Use TFPI2 antibody to detect expression changes during inflammation

  • In murine models, TFPI-2 expression increases 10-fold in spleen within 1 hour of LPS injection

  • Design time-course studies to capture expression dynamics across different tissues

• TFPI2 in bacterial infection models:

  • TFPI-2 knockout mice show susceptibility to Gram-negative bacterial infection

  • Methods to study:

    • Compare wild-type vs. TFPI-2 knockout mice response to infection

    • Use TFPI2 antibody to track expression in different tissues during infection

    • Perform immunohistochemistry on infected tissues

• Complement activation studies:

  • TFPI2 C-terminal peptides promote complement-mediated bacterial killing

  • Use antibodies against different TFPI2 domains to study which regions are involved

  • Develop assays to measure complement activation in the presence of TFPI2

• Clinical relevance in inflammatory conditions:

  • Analyze TFPI2 expression/fragments in patient samples

  • TFPI2 fragments have been detected in sputum from COPD patients

  • Correlate fragment patterns with disease severity or clinical outcomes

How do I design experiments to study the relationship between TFPI2 and the ERK signaling pathway?

To investigate TFPI2's role in ERK signaling:

• Expression manipulation studies:

  • Establish cell lines with:

    • TFPI2 overexpression

    • TFPI2 knockdown/knockout

    • Domain-specific TFPI2 mutants

  • Use TFPI2 antibody to confirm expression levels by Western blot

• Signaling pathway analysis:

  • Assess the phosphorylation status of ERK1/2 in response to TFPI2 manipulation

  • Research shows TFPI2 overexpression decreases phosphorylation of ERK1/2

  • Monitor translocation of p-ERK1/2 from cytoplasm to nucleus

  • Experimental design should include:

    • Time-course analysis after TFPI2 manipulation

    • Growth factor stimulation to activate ERK pathway

• Interaction studies:

  • Investigate if TFPI2 directly or indirectly affects ERK phosphorylation

  • Examine potential interactions with upstream components of ERK pathway

  • Use co-immunoprecipitation followed by Western blot analysis

• Functional readouts:

  • Assess changes in:

    • Cell proliferation (MTT assay)

    • Cell invasion (Transwell invasion assays)

    • Gene expression profiles (qRT-PCR of ERK target genes)

  • Determine if ERK pathway inhibitors can mimic TFPI2's effects

How can I troubleshoot non-specific binding or high background when using TFPI2 Antibody, HRP conjugated?

If experiencing high background or non-specific binding:

• Optimize blocking conditions:

  • Test different blocking agents (BSA, non-fat dry milk, normal serum)

  • Increase blocking time (1-2 hours at room temperature or overnight at 4°C)

  • Add 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

• Antibody dilution optimization:

  • Perform a dilution series to find optimal concentration

  • For HRP-conjugated antibodies, generally start with 1:500-1:2000 dilution

  • Higher dilutions may reduce background while maintaining specific signal

• Buffer modifications:

  • Add 0.1-0.5% Tween-20 to wash buffers

  • Consider adding 0.1-0.5M NaCl to reduce ionic interactions

  • For tissue sections, include 0.3% hydrogen peroxide treatment to block endogenous peroxidase activity

• Sample preparation improvements:

  • Ensure proper fixation and processing of samples

  • For frozen sections, allow complete drying before fixation

  • For cell lysates, ensure thorough removal of detergents through dialysis

• Control experiments:

  • Include no-primary antibody controls

  • Use isotype control antibodies at the same concentration

  • Pre-absorb antibody with recombinant TFPI2 protein

What are the best practices for long-term storage and handling of TFPI2 Antibody, HRP conjugated?

To maintain antibody performance over time:

• Storage conditions:

  • Store at -20°C or -80°C as recommended by manufacturer

  • Avoid repeated freeze-thaw cycles (more than 2-3 cycles)

  • Consider aliquoting into single-use volumes upon receipt

• Working dilution preparation:

  • Thaw antibody on ice

  • Prepare fresh working dilutions on the day of experiment

  • Do not store diluted antibody for extended periods

• HRP stability considerations:

  • Protect from light to prevent photobleaching of HRP

  • Avoid exposure to heavy metals and sodium azide, which inhibit HRP activity

  • Store in non-metal containers (plastic tubes recommended)

• Quality control:

  • Periodically validate antibody performance using positive control samples

  • Document lot numbers and maintain records of performance

  • Consider including a standard curve with each experiment to ensure consistent sensitivity

• Alternative storage methods:

  • For frequent use, consider storing small aliquots at 4°C with 0.02% sodium azide for up to 1 month

  • Note that sodium azide inhibits HRP and must be removed before use