Phospho-F3 (S290) Antibody

What is the Phospho-F3 (S290) antibody and what does it detect?

The Phospho-F3 (S290) antibody is a specialized antibody that specifically recognizes the human Tissue Factor (TF, also known as Coagulation Factor III or F3) when phosphorylated at the serine 290 residue. This antibody has been developed to detect post-translational modifications that may regulate TF function. The antibody is typically raised in rabbits as a polyclonal IgG and is purified using affinity chromatography with epitope-specific immunogens to ensure specificity for the phosphorylated form of the protein .

What are the common applications for Phospho-F3 (S290) antibody?

Phospho-F3 (S290) antibody has been validated for several key applications in molecular biology and immunology research:

• Western Blotting (WB): Typically used at dilutions of 1:500-1:2000

• Immunohistochemistry (IHC): Recommended dilutions range from 1:100-1:300

• Enzyme-Linked Immunosorbent Assay (ELISA): Often used at high dilutions up to 1:20000

These applications allow researchers to detect and quantify phosphorylated TF in various sample types including cell lysates, tissue sections, and purified protein preparations .

What is the structure and handling requirements for Phospho-F3 (S290) antibody?

The Phospho-F3 (S290) antibody is typically supplied in liquid form in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide for stability. For optimal performance, the antibody should be stored at -20°C for long-term storage (up to one year). For frequent use over shorter periods (up to one month), storage at 4°C is recommended. It's important to avoid repeated freeze-thaw cycles as this can degrade antibody quality and performance .

How should I design experiments to validate the specificity of Phospho-F3 (S290) antibody?

Validating phospho-specific antibodies requires careful experimental design to confirm both specificity and sensitivity. Based on established practices in phospho-antibody validation, researchers should:

• Perform blocking peptide experiments: Compare immunostaining with and without pre-incubation with the phosphorylated peptide immunogen. Complete blocking of signal with the phosphopeptide confirms specificity.

• Use phosphatase treatment controls: Treat one sample set with lambda phosphatase to remove phosphorylation and confirm loss of antibody reactivity.

• Include site-directed mutants: Test the antibody against S290A mutant constructs of F3/TF, which should show minimal or no reactivity if the antibody is truly phospho-specific.

• Test multiple applications: Validate specificity across Western blot, IHC and ELISA to ensure consistent performance .

Published validation data for phospho-TF (S290) antibodies typically include Western blot analysis of TNF-treated RAW264.7 cells, showing clear signal reduction when samples are pre-incubated with blocking phosphopeptides .

What is the recommended protocol for phospho-F3 detection in cell culture systems?

For optimal detection of phosphorylated TF in cell culture systems, follow this methodological approach:

• Cell stimulation: Treat cells with appropriate stimuli (e.g., TNF at 20ng/ml for 30 minutes) to induce TF phosphorylation.

• Sample preparation:

  • Harvest cells in phospho-preserving lysis buffer containing phosphatase inhibitors

  • Include protease inhibitors (e.g., PMSF, complete protease inhibitor cocktail)

  • Maintain samples at 4°C throughout processing

• Western blotting protocol:

  • Use 7.5-12% SDS-PAGE gels for optimal separation

  • Include phosphatase-treated controls

  • Block membranes with 3-5% BSA (not milk, which contains phosphoproteins)

  • Incubate with phospho-F3 (S290) antibody at 1:1000 dilution overnight at 4°C

  • Wash thoroughly and detect with appropriate secondary antibodies

• Signal verification: Always run parallel blots for total F3/TF to calculate phosphorylation ratios properly .

What protocol should be followed for phosphoprotein detection in human samples?

When working with human samples such as PBMCs or tissue specimens, specialized protocols for phosphoprotein preservation are essential:

• Sample preparation and fixation:

  • Process samples immediately upon collection

  • Fix cells with pre-warmed Cytofix Buffer (equal volume) at 37°C for 10-12 minutes

  • Centrifuge at 600g for 6-8 minutes

• Permeabilization:

  • Disrupt cell pellet by gentle vortexing

  • Add 1mL of Perm/Wash Buffer I per 1-10 × 10^6 cells

  • Incubate for 15-30 minutes at room temperature

• Antibody staining:

  • Resuspend cells at 5-10 × 10^6 cells/mL in Perm/Wash Buffer

  • Incubate with phospho-F3 (S290) antibody for 60 minutes at room temperature, protected from light

  • Wash thoroughly with Perm/Wash Buffer

• Analysis:

  • For flow cytometry: Resuspend in appropriate buffer at 0.5-1 × 10^6 cells/500μL

  • For imaging: Mount samples using phospho-preserving mounting media

This protocol maintains phosphorylation status while ensuring accessibility of the antibody to its target epitope .

How does phosphorylation at S290 affect TF function in coagulation cascades?

Phosphorylation of TF at S290 represents a significant post-translational regulatory mechanism for coagulation activity. Research data indicates that:

• Signaling pathway involvement: S290 phosphorylation occurs downstream of inflammatory signaling, particularly after TNF-α stimulation of cells expressing TF.

• Functional consequences: Phosphorylation at this site may modulate:

  • TF procoagulant activity

  • Interaction with Factor VII/VIIa

  • Cell surface exposure of TF

  • Partitioning of TF into lipid rafts

• Regulatory mechanisms: The phosphorylation state of S290 appears to be dynamically regulated, with both kinase and phosphatase activities controlling the equilibrium.

Further research is needed to fully elucidate the complete signaling pathways that regulate TF phosphorylation at S290 and the precise mechanisms by which this modification alters TF function in different cellular contexts .

What controls should be included when investigating F3/TF phosphorylation interactions with other proteins?

When investigating how F3/TF phosphorylation affects protein-protein interactions, researchers should include several critical controls:

• Phospho-mimetic and phospho-deficient mutants: Generate S290E (phospho-mimetic) and S290A (phospho-deficient) TF mutants to compare with wild-type protein.

• Kinase inhibitors: Include appropriate kinase inhibitors to prevent phosphorylation as negative controls.

• Phosphatase treatments: Treat samples with lambda phosphatase to remove phosphorylation and observe effects on protein interactions.

• Verification of phosphorylation status: Always verify phosphorylation status using the phospho-F3 (S290) antibody alongside co-immunoprecipitation experiments.

• Multiple detection methods: Confirm protein interactions using at least two different techniques (e.g., co-IP, proximity ligation assay, FRET).

These controls help distinguish between phosphorylation-dependent and -independent interactions and provide robust validation of experimental findings .

How can phospho-F3 (S290) antibody be used to investigate heat shock protein interactions?

Emerging research indicates potential connections between TF phosphorylation and heat shock protein (HSP) pathways. To investigate these interactions:

• Co-immunoprecipitation assays:

  • Immunoprecipitate with phospho-F3 (S290) antibody followed by Western blotting for HSP90 and CDC37

  • Alternatively, immunoprecipitate with HSP90 antibodies and probe for phospho-F3

• Proximity ligation assays:

  • Use paired antibodies against phospho-F3 (S290) and HSP90/CDC37 to visualize in situ interactions

  • Quantify interaction signals under different cellular conditions

• Inhibitor studies:

  • Treat cells with HSP90 inhibitors (e.g., geldanamycin, onalespib, 17-AAG) to assess effects on TF phosphorylation

  • Monitor both phosphorylation status and protein stability

• Mutational analysis:

  • Compare wild-type TF with phospho-mimetic and phospho-deficient mutants for HSP interaction

This approach can reveal whether HSP90 chaperone systems regulate TF phosphorylation, stability, or function, similar to their roles with other client proteins .

Why might I observe inconsistent results when detecting phospho-F3 (S290) in different sample types?

Inconsistent results when detecting phospho-F3 (S290) can stem from several methodological factors:

• Phosphorylation preservation issues:

  • Rapid dephosphorylation during sample handling

  • Inadequate phosphatase inhibitor cocktails

  • Temperature fluctuations during processing

• Sample-specific challenges:

  • Cell/tissue-specific phosphatase activity levels

  • Varying expression levels of total F3/TF

  • Different phosphorylation kinetics in various cell types

• Technical considerations:

  • Antibody lot-to-lot variations

  • Buffer incompatibilities

  • Blocking reagent interference (avoid milk-based blockers)

To overcome these challenges, implement:

• Immediate sample processing

• Comprehensive phosphatase inhibitor cocktails (e.g., PhosSTOP)

• Standardized positive controls for each experiment

• Consistent temperature control throughout processing .

How can I optimize phospho-F3 (S290) antibody performance in challenging applications?

For optimal performance in challenging applications such as low-abundance samples or difficult tissue types:

• Signal amplification strategies:

  • Consider tyramide signal amplification (TSA) for IHC applications

  • Use high-sensitivity ECL reagents for Western blots

  • Implement biotin-streptavidin systems for ELISA

• Sample enrichment:

  • Perform phosphoprotein enrichment using TiO₂ or IMAC columns

  • Consider immunoprecipitation to concentrate target proteins before analysis

• Antibody optimization:

  • Test extended incubation times (overnight at 4°C)

  • Optimize antibody concentration with titration experiments

  • Consider alternative detection systems (fluorescent vs. chromogenic)

• Buffer modifications:

  • Adjust detergent concentrations to reduce background

  • Modify salt concentrations to enhance specificity

  • Add protein carriers to prevent non-specific binding

These approaches can significantly improve signal-to-noise ratios and enhance detection of low-abundance phosphorylated forms of TF .

How can I distinguish between specific phospho-F3 (S290) signal and cross-reactivity with other phosphoproteins?

Distinguishing true phospho-F3 (S290) signal from potential cross-reactivity requires rigorous validation:

• Comprehensive specificity testing:

  • Test antibody against multiple phosphorylated proteins/peptides

  • Include S290A mutant controls

  • Perform peptide competition assays with both specific and non-specific phosphopeptides

• Advanced validation approaches:

  • Perform phosphoproteomics analysis of immunoprecipitated material

  • Use knockout/knockdown models to confirm signal absence

  • Employ orthogonal detection methods with different antibody clones

• Quantitative assessments:

  • Calculate signal ratios in the presence and absence of competing peptides

  • Perform dose-response curves with phosphatase treatments

  • Compare signal patterns across multiple antibodies targeting different epitopes

These strategies help establish confidence in signal specificity and minimize misinterpretation of results due to antibody cross-reactivity .

How does phosphorylation at S290 compare with other post-translational modifications of F3/TF?

F3/TF undergoes multiple post-translational modifications that collectively regulate its function. Comparing S290 phosphorylation with other modifications:

• Other phosphorylation sites:

  • While S290 phosphorylation appears to modulate coagulation activity, other phosphorylation sites may affect different aspects of TF function

  • Phosphorylation at multiple sites may have synergistic or antagonistic effects

• Glycosylation interactions:

  • Research indicates that glycosylation affects TF folding and surface expression

  • S290 phosphorylation may interact with glycosylation status to fine-tune TF activity

• Ubiquitination and degradation:

  • Phosphorylation often serves as a recognition signal for ubiquitin ligases

  • S290 phosphorylation may influence TF protein stability and turnover

• Functional hierarchy:

  • Different modifications may predominate under specific cellular conditions

  • Temporal dynamics of modifications likely create complex regulatory patterns

This multi-dimensional regulation highlights the importance of studying modifications in combination rather than isolation .

What methodological approaches can reveal the temporal dynamics of F3/TF phosphorylation?

Understanding the temporal dynamics of F3/TF phosphorylation requires specialized approaches:

• Time-course experiments:

  • Design stimulation experiments with multiple time points

  • Use phosphatase inhibitors to "freeze" phosphorylation states

  • Analyze both total and phosphorylated TF at each time point

• Pulse-chase approaches:

  • Combine metabolic labeling with phospho-specific immunoprecipitation

  • Track newly synthesized vs. existing protein pools

  • Monitor phosphorylation kinetics during protein maturation

• Live-cell imaging techniques:

  • Develop phospho-specific biosensors based on FRET principles

  • Use split fluorescent protein systems coupled to phospho-binding domains

  • Implement optogenetic tools to trigger phosphorylation with temporal precision

• Mathematical modeling:

  • Integrate experimental data into kinetic models of phosphorylation/dephosphorylation

  • Predict system behavior under varying conditions

  • Validate model predictions with targeted experiments

These approaches provide insights into the dynamic regulation of TF phosphorylation in response to various stimuli and cellular conditions .

How might phospho-F3 (S290) antibodies advance understanding of TF in disease pathophysiology?

Phospho-F3 (S290) antibodies represent important tools for investigating TF in various pathological conditions:

• Cancer research applications:

  • Analyze phospho-TF status in tumor samples versus normal tissues

  • Correlate phosphorylation patterns with tumor aggressiveness and metastatic potential

  • Investigate connections between TF phosphorylation and cancer-associated thrombosis

• Cardiovascular disease contexts:

  • Examine phospho-TF in atherosclerotic plaques

  • Study how inflammatory signals modulate TF phosphorylation in vascular cells

  • Correlate phosphorylation status with plaque stability and thrombogenicity

• Inflammatory disorders:

  • Investigate phospho-TF regulation in autoimmune diseases

  • Study how different inflammatory mediators affect TF phosphorylation patterns

  • Develop therapeutic strategies targeting phosphorylation-specific activities

• Biomarker development:

  • Evaluate phospho-TF as a potential biomarker for thrombotic risk

  • Develop assays to detect circulating phospho-TF in patient samples

  • Correlate phospho-TF levels with disease progression and therapeutic responses

These applications could provide new insights into disease mechanisms and potential therapeutic approaches targeting specific phosphorylated forms of TF .