TNS4 Antibody, FITC conjugated

What is TNS4 and why is it an important research target?

TNS4 (Tensin-4, also known as CTEN) is a focal adhesion protein that directly interacts with phosphorylated receptor tyrosine kinases, particularly MET, via its SH2 domain to positively regulate cell survival, proliferation, and migration. Unlike other tensin family members such as TNS3, TNS4 lacks an actin-binding domain and has been suggested to possess oncogenic functions in multiple cancer types .

TNS4 has gained significant research interest because:

• It is significantly upregulated in colorectal, lung, ovarian, gastric, and head and neck cancers with concomitant downregulation of TNS3 levels

• It forms complexes with MET and β1-integrin that regulate cell migration

• It stabilizes MET by inhibiting its endocytosis and subsequent lysosomal degradation

• It promotes EGF-induced cell migration by displacing TNS3 from integrin ITGB1

• It suppresses ligand-induced degradation of EGFR by reducing EGFR ubiquitination

How does FITC conjugation affect antibody functionality in TNS4 research applications?

• Conjugation chemistry: FITC covalently couples primarily to the epsilon-amino group of lysine residues of surface glycoproteins in antibodies . This modification may alter antibody binding characteristics if lysine residues are located within or near the antigen-binding site.

• Fluorescence parameters: FITC-conjugated antibodies have an absorption maximum at 492nm and emission maximum at 520nm , providing a bright green fluorescence suitable for multicolor applications.

• Molecular F/P (fluorescein/protein) ratio: Optimal labeling is critical, as under-labeling reduces sensitivity while over-labeling can compromise antibody binding capacity. Research indicates that optimal conjugation occurs at pH 9.5 with an initial protein concentration of 25 mg/ml and reaction times of 30-60 minutes at room temperature .

• Storage considerations: FITC-conjugated antibodies typically require storage at -20°C with glycerol and protection from light to prevent photobleaching .

What are the validated applications for TNS4 antibody, FITC conjugated?

Based on the available research data, TNS4 antibody, FITC conjugated has been validated for the following applications:

For optimal results, validated dilution ranges typically include 1:20-1:100 for IF or Flow cytometry and 1:1000-1:5000 for WB applications .

How should I design experiments to study TNS4-MET-integrin interactions using FITC-conjugated antibodies?

To effectively study TNS4-MET-integrin interactions using FITC-conjugated TNS4 antibodies, consider this comprehensive experimental approach:

• Co-localization studies:

  • Perform triple immunofluorescence staining with TNS4 antibody-FITC, anti-MET, and anti-β1-integrin (with distinct fluorophores)

  • Use high-resolution confocal microscopy to visualize potential co-localization, particularly in adhesion sites as reported by Muharram et al.

  • Include both unstimulated cells and cells stimulated with HGF (which increases MET-TNS4 co-localization)

• Co-immunoprecipitation validation:

  • Use TNS4 antibody for immunoprecipitation followed by Western blot analysis for MET and β1-integrin

  • Compare results in control cells vs. HGF-stimulated cells (HGF increases recruitment of MET to TNS4)

  • Include suitable controls: IgG control, lysate input, reverse co-IP

• Functional studies:

  • Generate TNS4 knockdown cells (using siRNA) and TNS4-overexpressing cells

  • Reconstitute with wild-type TNS4 or the TNS4 MET-binding mutant (TNS4_R474A)

  • Assess cell migration using cell-derived matrices with and without HGF stimulation

  • Monitor internalization of cell-surface receptors using biotinylation-based endocytosis assays

• Binding domain characterization:

  • Generate recombinant proteins containing specific TNS4 domains (SH2-PTB fragment)

  • Perform pull-down assays with cell lysates from HGF-responsive cells (e.g., A549) and MET-amplified cells (e.g., GTL-16)

  • Compare binding with wild-type versus mutant constructs (e.g., R474A mutation in the SH2 domain)

This multi-faceted approach will provide robust evidence for the functional relationships between TNS4, MET, and integrins in cellular physiology and potential pathological contexts.

What controls are essential when using TNS4 antibody-FITC in flow cytometry for analyzing T cell responses?

When using TNS4 antibody-FITC in flow cytometry for analyzing T cell responses, the following essential controls should be implemented to ensure valid and reproducible results:

• Antibody specificity controls:

  • Isotype control: Include a FITC-conjugated isotype-matched irrelevant antibody at the same concentration to assess non-specific binding

  • Blocking control: Pre-incubate cells with unconjugated TNS4 antibody before staining with TNS4-FITC to confirm specificity

  • TNS4 knockout/knockdown cells: If available, use cells where TNS4 has been genetically deleted or suppressed

• Fluorescence controls:

  • Unstained cells: To establish autofluorescence baseline

  • Single-color controls: For compensation when using multiple fluorophores

  • Fluorescence-minus-one (FMO) control: Include all antibodies except TNS4-FITC to define the FITC-negative population boundary

• Experimental design controls:

  • Positive control: Include samples known to express TNS4 (e.g., cancer cell lines with documented TNS4 expression)

  • Negative control: Include samples known not to express TNS4

  • Biological replicates: At least three independent experiments

  • Technical replicates: Multiple samples from the same biological source

• T cell-specific controls:

  • T cell activation markers: Include markers like CD69, CD137, and OX40 as used in activation-induced marker (AIM) assays for T cell responses

  • Non-T cell controls: Include other leukocyte populations to assess lineage-specific expression

  • Antigen-specific stimulation controls: Compare unstimulated vs. antigen-stimulated T cells, as in peptide pool stimulation protocols

• Data analysis quality controls:

  • Viability dye: Include a viability dye such as LIVE/DEAD™ Fixable Near-IR to exclude dead cells

  • Doublet discrimination: Use forward scatter height vs. area to exclude cell aggregates

  • Consistent gating strategy: Define and document a consistent gating approach

These comprehensive controls will ensure that any observed changes in TNS4 expression in T cells are genuine and biologically meaningful rather than technical artifacts.

How should I optimize the conjugation ratio when preparing custom TNS4 antibody-FITC conjugates for research applications?

Optimizing the conjugation ratio when preparing custom TNS4 antibody-FITC conjugates requires a systematic approach to achieve maximum fluorescence without compromising antibody activity:

• Determine optimal reaction conditions:

  • pH: Use carbonate-bicarbonate buffer at pH 9.5, which research has shown to be optimal for FITC conjugation

  • Protein concentration: Begin with an antibody concentration of 25 mg/ml, as this has been demonstrated to achieve maximal labeling in a short time

  • Reaction time: Aim for 30-60 minutes at room temperature, which typically provides optimal conjugation without excessive labeling

  • FITC quality: Use high-purity FITC to ensure consistent conjugation efficiency

• Test multiple F/P (fluorescein/protein) ratios:

  • Prepare a range of conjugates with varying FITC:antibody molar ratios (typically 5:1 to 20:1)

  • Measure the actual F/P ratio spectrophotometrically using the formula:
    F/P ratio = (A495 × MW of antibody) / (195 × antibody concentration in mg/ml)

  • Target F/P ratios between 3-8 moles FITC per mole IgG; commercial preparations typically achieve around 3.1 moles FITC per mole IgG

• Separate optimally labeled antibodies:

  • Use gradient DEAE Sephadex chromatography to separate under-labeled, optimally labeled, and over-labeled fractions

  • Collect fractions and determine F/P ratios for each

  • Pool fractions with optimal F/P ratios (typically 3-6)

• Validate conjugate functionality:

  • Assess binding activity using flow cytometry or immunofluorescence with cells known to express TNS4

  • Compare the custom conjugate to commercial TNS4-FITC antibodies if available

  • Perform parallel assays with unconjugated TNS4 antibody to ensure conjugation hasn't significantly impaired binding capability

  • Validate specificity using TNS4 knockdown/knockout samples

• Stability testing:

  • Assess storage stability at -20°C with different stabilizers (e.g., 50% glycerol, BSA at 5 mg/ml)

  • Test freeze-thaw stability by subjecting aliquots to multiple freeze-thaw cycles

  • Evaluate photostability by measuring fluorescence intensity after repeated light exposure

• Quantitative assessment matrix:

Following this systematic approach will ensure the production of TNS4 antibody-FITC conjugates with optimal performance characteristics for your specific research applications.

How can TNS4 antibody-FITC be effectively used to study MET receptor trafficking in cancer cells?

TNS4 antibody-FITC can be effectively employed to investigate MET receptor trafficking in cancer cells through several sophisticated methodological approaches:

• Live-cell imaging of TNS4-MET dynamics:

  • Transfect cells with MET-RFP (or another compatible fluorophore)

  • Use TNS4-FITC antibody with cell-permeable delivery systems (e.g., protein transfection reagents)

  • Perform time-lapse confocal microscopy to track co-localization patterns during:

    • Basal conditions

    • HGF stimulation (which increases MET-TNS4 co-localization)

    • Treatment with MET kinase inhibitors (which disrupts TNS4-MET association)

• Quantitative endocytosis and recycling assays:

  • Implement cell-surface biotinylation-based endocytosis assays as described by Muharram et al. :

    • Surface-biotinylate cells to label cell-surface proteins

    • Allow endocytosis to occur for various time periods

    • Remove remaining surface biotin with a membrane-impermeable reducing agent

    • Immunoprecipitate internalized biotinylated proteins and detect MET by Western blotting

  • Use TNS4-FITC in parallel flow cytometry assays to correlate TNS4 expression with MET internalization rates

  • Compare results between:

    • Control cells

    • TNS4-silenced cells (which show increased MET endocytosis)

    • TNS4-overexpressing cells (which show decreased MET endocytosis)

    • Cells expressing the TNS4 MET-binding mutant (TNS4_R474A)

• Pulse-chase immunofluorescence approach:

  • Label cell-surface MET with a non-FITC primary antibody at 4°C

  • Allow internalization at 37°C for various time points

  • Fix cells and stain with TNS4-FITC antibody

  • Analyze co-localization of MET with TNS4 during internalization process

  • Counterstain with markers for different endocytic compartments (early endosomes, late endosomes, lysosomes)

• FACS-based MET internalization kinetics:

  • Utilize the antibody labeling and FACS analysis approach described in Muharram et al. :

    • Label cell-surface MET with a specific antibody

    • Allow internalization for various time periods

    • Quantify remaining surface MET by flow cytometry

  • Correlate surface MET levels with TNS4 expression measured by TNS4-FITC

• Super-resolution microscopy for detailed localization:

  • Employ techniques like STORM or STED microscopy for nanoscale resolution

  • Determine precise spatial relationships between TNS4 and MET at:

    • Cell membrane

    • Adhesion sites where TNS4, MET, and β1-integrin have been shown to co-localize

    • Intracellular trafficking compartments

This comprehensive approach will provide valuable insights into how TNS4 regulates MET receptor trafficking, particularly its role in stabilizing MET by inhibiting endocytosis and subsequent degradation, which promotes cancer cell survival, proliferation, and migration.

What methodological approaches can address the issue of photobleaching when using TNS4 antibody-FITC in long-term live cell imaging?

Addressing photobleaching of TNS4 antibody-FITC during long-term live cell imaging requires a multi-faceted approach combining optical, chemical, and computational strategies:

• Optical and microscopy optimizations:

  • Minimize excitation intensity: Use the minimum laser power or lamp intensity required for adequate signal detection

  • Implement pulsed illumination: Use triggered or time-gated illumination to expose the sample only during image acquisition

  • Employ spinning disk confocal microscopy: This reduces photobleaching compared to traditional point-scanning confocal systems

  • Use oxygen-scavenging objectives: Objectives designed to reduce oxygen diffusion at the sample interface

  • Optimize detection sensitivity: Use high-quantum efficiency cameras and photomultiplier tubes to detect signal with minimal excitation

• Chemical approaches:

  • Use anti-fade agents in imaging media:

    • ProLong™ Live Antifade Reagent for live-cell applications

    • Vitamin C (ascorbic acid) at 1-10 mM concentration

    • Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) at 1-2 mM

  • Incorporate oxygen scavenging systems:

    • Glucose oxidase/catalase (GLOX) system

    • Protocatechuic acid (PCA)/protocatechuate-3,4-dioxygenase (PCD) system

  • Apply phytochemical protective agents:

    • Cyclooctatetraene (COT)

    • 4-nitrobenzyl alcohol (NBA)

    • Methylviologen

• Alternative labeling strategies:

  • Use TNS4 fused to fluorescent proteins: Generate stable cell lines expressing TNS4-GFP or TNS4-mEmerald

  • Employ photoconvertible fluorescent proteins: TNS4 fused to proteins like mEos or Dendra2

  • Implement SNAP or Halo tag technology: Tag TNS4 with SNAP/Halo tags and use cell-permeable fluorescent ligands for renewable labeling

  • Consider quantum dot conjugation: TNS4 antibody conjugated to quantum dots exhibits superior photostability

• Image acquisition and processing techniques:

  • Implement time-interval imaging: Capture frames at defined intervals rather than continuously

  • Use denoising algorithms: Apply computational methods to extract signal from noisy, low-light images

  • Employ photobleaching correction algorithms:

    • Exponential decay correction

    • Reference-based correction using internal standards

    • Machine learning-based restoration

  • Implement Bayesian statistical frameworks to extract maximal information from minimal photon counts

• Experimental design considerations:

  • Design intermittent labeling protocols: Periodically introduce fresh TNS4-FITC antibody during long-term experiments

  • Use photoactivation microscopy: Label the entire population but activate only a subset for each time point

  • Implement correlative microscopy: Combine live fluorescence imaging with endpoint super-resolution techniques

By systematically implementing these strategies, researchers can significantly extend the useful imaging time of TNS4 antibody-FITC in live cell applications while maintaining adequate signal-to-noise ratios for reliable scientific interpretation.

How can TNS4 antibody-FITC be used to investigate TNS4's role in hypoxia-driven cancer progression?

To investigate TNS4's role in hypoxia-driven cancer progression using TNS4 antibody-FITC, researchers can implement the following comprehensive methodological approaches:

• Hypoxia-responsive TNS4 expression profiling:

  • Establish in vitro hypoxia models using:

    • Hypoxic chambers (1-5% O₂) for varying durations

    • Hypoxia-mimetic agents (CoCl₂, DMOG, or DFO)

  • Quantify TNS4 expression changes using:

    • Flow cytometry with TNS4 antibody-FITC to measure protein expression levels across cell populations

    • Immunofluorescence microscopy to assess subcellular localization changes

  • Correlate with HIF-1α expression, as research has shown HIF-1α transcriptionally regulates TNS4 expression

• Mechanistic analysis of hypoxia-TNS4-integrin axis:

  • Implement multicolor flow cytometry and immunofluorescence using:

    • TNS4 antibody-FITC

    • Anti-integrin α5β1 antibodies with compatible fluorophores

    • HIF-1α antibodies

  • Analyze the effect of hypoxia on:

    • TNS4-integrin α5β1 complex formation

    • FAK activation status using phospho-FAK antibodies

    • PI3K/Akt pathway activation

  • Include parallel experiments with:

    • HIF-1α inhibitors (e.g., acriflavine, YC-1)

    • HIF-1α knockdown/knockout

    • TNS4 knockdown/overexpression

• 3D tumor spheroid models:

  • Generate tumor spheroids from cancer cell lines with native or manipulated TNS4 levels

  • Create natural hypoxic gradients within spheroids (core becomes hypoxic)

  • Perform live imaging with TNS4 antibody-FITC (for surface cells) or fixed sectioning for whole-spheroid analysis

  • Correlate TNS4 expression with:

    • Distance from spheroid surface (oxygen gradient)

    • Cell proliferation markers

    • Cell invasion capacity

    • HIF-1α expression

• Patient-derived xenograft (PDX) analysis:

  • Implant patient-derived tumor samples in immunodeficient mice

  • Monitor growth with varying oxygen conditions

  • Harvest tumors and analyze sections using TNS4 antibody-FITC to correlate TNS4 expression with:

    • Hypoxic regions (identified using pimonidazole staining)

    • Tumor invasiveness

    • Metastatic potential

    • HIF-1α expression

• High-resolution intravital imaging:

  • Develop window chamber models in mice bearing tumors

  • Inject TNS4 antibody-FITC intravenously

  • Perform real-time confocal microscopy to visualize:

    • TNS4 expression in relation to tumor vasculature

    • TNS4 dynamics in hypoxic vs. normoxic tumor regions

    • Cell migration patterns in relation to TNS4 expression

• Transcriptional regulation analysis:

  • Implement chromatin immunoprecipitation (ChIP) assays to confirm HIF-1α binding to the TNS4 promoter

  • Use reporter assays with TNS4 promoter constructs under hypoxic conditions

  • Perform site-directed mutagenesis of potential HIF-binding elements in the TNS4 promoter

• Therapeutic targeting assessment:

  • Test TNS4-targeting strategies in hypoxic conditions:

    • siRNA/shRNA against TNS4

    • Small molecule inhibitors of TNS4-MET or TNS4-integrin interactions

  • Monitor effects on:

    • Cell survival

    • Metastatic potential

    • Response to chemotherapy

    • Radiation sensitivity

This comprehensive approach leverages the sensitivity and specificity of TNS4 antibody-FITC to thoroughly investigate the role of TNS4 in hypoxia-driven cancer progression, potentially identifying novel therapeutic targets and strategies.

How should I interpret discrepancies between TNS4 antibody-FITC staining patterns and TNS4-GFP fusion protein localization?

When encountering discrepancies between TNS4 antibody-FITC staining patterns and TNS4-GFP fusion protein localization, a systematic analytical approach is necessary to determine the true biological significance:

• Analyze potential sources of technical artifacts:

  • Antibody-related factors:

    • Epitope accessibility: Determine if the TNS4 antibody epitope might be masked in certain subcellular contexts

    • Antibody specificity: Validate using Western blot of lysates from TNS4-GFP expressing cells to confirm recognition of both endogenous and GFP-tagged TNS4

    • Cross-reactivity: Test the antibody on TNS4 knockout/knockdown cells to assess potential non-specific binding

  • GFP fusion protein considerations:

    • GFP interference: The GFP tag (27 kDa) might disrupt protein folding, interactions, or localization

    • Overexpression artifacts: TNS4-GFP overexpression may saturate binding sites or overwhelm regulatory mechanisms

    • Fusion position effects: N- vs. C-terminal GFP tagging may differently affect TNS4 function and localization

• Implement dual-detection strategies:

  • Perform co-staining experiments:

    • Express TNS4-GFP and stain with TNS4 antibody-FITC (use anti-GFP antibody with different fluorophore)

    • Analyze overlap and divergence patterns quantitatively using colocalization coefficients

    • Include appropriate controls for bleed-through and cross-detection

  • Validate with orthogonal approaches:

    • Use TNS4 antibodies targeting different epitopes

    • Implement alternative tags (e.g., FLAG, Myc) and compare localization patterns

    • Apply proximity ligation assays (PLA) to detect protein-protein interactions

• Conduct functional validation experiments:

  • Mutational analysis:

    • Generate TNS4 mutants affecting key domains (SH2, PTB) in both antibody-detected and GFP-fusion contexts

    • Assess if localization changes parallel functional impacts (e.g., R474A mutation disrupting MET binding)

    • Examine if discrepancies persist across different mutant forms

  • Stimulus response analysis:

    • Compare localization changes upon HGF stimulation (which promotes TNS4-MET interaction)

    • Assess response to FAK inhibitors, which should affect TNS4-mediated signaling

    • Determine if discrepancies are consistent or condition-dependent

• Organelle-specific colocalization assessment:

  • Systematically compare TNS4 antibody-FITC and TNS4-GFP colocalization with markers for:

    • Focal adhesions (paxillin, vinculin)

    • Cell membrane and membrane subdomains

    • Vesicular trafficking compartments (early/late endosomes, lysosomes)

    • ER, Golgi, and secretory pathway components

  • Quantify overlap using:

    • Pearson's correlation coefficient

    • Manders' colocalization coefficients

    • Distance-based methods (e.g., nearest neighbor analysis)

• Resolution-dependent analysis:

  • Employ super-resolution microscopy techniques:

    • STED, STORM, or PALM imaging to resolve nano-scale distribution differences

    • Single-molecule tracking to assess dynamic behavior differences

    • Correlative light-electron microscopy for ultrastructural context

• Interpretation framework matrix:

By systematically addressing these considerations, researchers can determine whether discrepancies represent technical artifacts or biologically meaningful insights into TNS4 function and regulation.

What are the common pitfalls in analyzing TNS4 expression in immune cells using FITC-conjugated antibodies?

Analyzing TNS4 expression in immune cells using FITC-conjugated antibodies presents several methodological challenges that researchers should anticipate and address:

• Autofluorescence interference:

  • Challenge: Immune cells, particularly activated and phagocytic cells, exhibit significant autofluorescence in the FITC emission spectrum.

  • Solution:

    • Implement strict autofluorescence controls for each cell type and activation state

    • Consider alternative fluorophores with longer emission wavelengths for high-autofluorescence samples

    • Apply spectral unmixing algorithms to separate FITC signal from autofluorescence

    • Pre-treat samples with quenching agents such as Sudan Black B or TrueBlack™

• Non-specific binding in activated immune cells:

  • Challenge: Activated T cells, B cells, and myeloid cells upregulate Fc receptors that can bind antibodies non-specifically.

  • Solution:

    • Use Fc receptor blocking reagents before antibody staining

    • Include isotype controls matched to TNS4 antibody-FITC concentration

    • Compare staining patterns between resting and activated cells

    • Validate specificity using TNS4 knockdown approaches

• TNS4 expression level variability across immune cell subsets:

  • Challenge: Baseline TNS4 expression may vary dramatically across lymphocyte, monocyte, and granulocyte populations.

  • Solution:

    • Perform comprehensive immune cell subset analysis using lineage markers

    • Establish baseline expression profiles for each major immune cell type

    • Implement fluorescence-minus-one (FMO) controls for accurate gating

    • Use standardized beads to calibrate fluorescence intensity measurements

• Context-dependent expression during immune responses:

  • Challenge: TNS4 expression may fluctuate during T cell activation, as seen with other proteins in activation-induced marker (AIM) assays .

  • Solution:

    • Design time-course experiments to capture expression dynamics

    • Compare TNS4 expression with established activation markers (CD69, CD137, OX40)

    • Analyze in context of both antigen-specific and non-specific stimulation

    • Examine expression in memory vs. naïve T cell populations

• FITC spectral limitations in multicolor panels:

  • Challenge: FITC has relatively broad emission spectrum that can overlap with other fluorophores.

  • Solution:

    • Carefully design multicolor panels with minimal spectral overlap

    • Perform comprehensive compensation using single-color controls

    • Consider using TNS4 antibodies conjugated to alternative fluorophores for complex panels

    • Implement advanced flow cytometry techniques such as spectral cytometry

• Data interpretation challenges in heterogeneous samples:

  • Challenge: Mixed cell populations may show bimodal or complex TNS4 expression patterns.

  • Solution:

    • Implement hierarchical gating strategies to isolate specific cell subpopulations

    • Use dimensionality reduction techniques (e.g., tSNE, UMAP) to visualize high-dimensional data

    • Combine flow cytometry with cell sorting and functional assays

    • Correlate TNS4 expression with functional outcomes in sorted populations

• Fixation and permeabilization effects:

  • Challenge: Different fixation protocols can affect FITC fluorescence and TNS4 epitope accessibility.

  • Solution:

    • Compare multiple fixation/permeabilization protocols

    • Optimize protocols for specific cell types

    • Consider live-cell staining for surface epitopes

    • Include protocol-matched positive controls

• Experimental validation matrix:

By addressing these methodological considerations, researchers can generate more reliable and interpretable data on TNS4 expression in immune cells using FITC-conjugated antibodies.

What approaches can resolve contradictory data between FITC-based flow cytometry and Western blot analysis of TNS4 expression?

When faced with contradictory results between FITC-based flow cytometry and Western blot analysis of TNS4 expression, researchers should implement a systematic troubleshooting strategy to reconcile these discrepancies:

• Analytical comparison of detection methodologies:

  • Epitope accessibility differences:

    • Flow cytometry: Detects surface-exposed or accessible epitopes in native conformation

    • Western blot: Detects denatured epitopes that may be masked in native proteins

    • Solution: Use multiple TNS4 antibodies targeting different epitopes in both techniques

  • Sensitivity threshold disparities:

    • Flow cytometry: Single-cell resolution with detection limits of ~500-1,000 molecules/cell

    • Western blot: Population average with detection limits often requiring 10-50 ng of target protein

    • Solution: Implement quantitative Western blotting with standard curves using recombinant TNS4

  • Post-translational modification impact:

    • Flow cytometry may detect specific TNS4 forms that are differentially recognized in Western blot

    • Solution: Use phosphorylation-specific antibodies or treatments (phosphatase, glycosidase) to assess modification effects

• Technical validation experiments:

  • Antibody cross-reactivity assessment:

    • Perform immunoprecipitation with TNS4 antibody followed by mass spectrometry

    • Test antibody on TNS4 knockout/knockdown samples in both flow cytometry and Western blot

    • Conduct peptide competition assays with the immunizing peptide

  • Sample preparation comparison:

    • Extract proteins using identical lysis buffers for both techniques

    • Compare fresh vs. fixed samples in flow cytometry

    • Assess effects of different detergents on epitope accessibility

  • Subcellular fractionation analysis:

    • Separate membrane, cytosolic, and nuclear fractions

    • Analyze each fraction by both Western blot and flow cytometry (after gentle permeabilization)

    • Determine if discrepancies are compartment-specific

• Biological validation strategies:

  • Expression modulation:

    • Generate cells with titratable TNS4 expression (e.g., inducible systems)

    • Compare dose-response curves between flow cytometry and Western blot

    • Assess linearity of detection across a range of expression levels

  • Context-dependent expression analysis:

    • Compare TNS4 detection across different cell states:

      • Basal vs. HGF-stimulated (which increases TNS4 levels)

      • Normoxic vs. hypoxic conditions (as HIF-1α regulates TNS4)

      • Different cell cycle phases

    • Determine if discrepancies persist across all conditions

  • Functional correlation studies:

    • Sort cells based on TNS4-FITC intensity by FACS

    • Analyze sorted populations by Western blot

    • Perform functional assays (migration, proliferation) on sorted populations

• Advanced reconciliation approaches:

  • Single-cell Western blot:

    • Implement microfluidic-based single-cell Western blot technology

    • Compare directly with flow cytometry data on a single-cell level

    • Assess if population averages mask subpopulation effects

  • Imaging flow cytometry:

    • Combine flow cytometry with microscopy using ImageStream technology

    • Visualize subcellular localization while quantifying expression

    • Correlate with conventional Western blot data

  • Alternative protein quantification methods:

    • Mass cytometry (CyTOF) with metal-conjugated TNS4 antibodies

    • Proximity extension assays or proximity ligation assays

    • ELISA or other immunoassays with defined detection thresholds

• Interpretation framework:

By systematically addressing these potential sources of discrepancy, researchers can reconcile contradictory data and gain deeper insight into the biology of TNS4 expression and regulation.

How can TNS4 antibody-FITC be used to investigate the bidirectional regulatory relationship between TNS4 and MET in cancer progression?

To investigate the bidirectional regulatory relationship between TNS4 and MET in cancer progression using TNS4 antibody-FITC, researchers can implement the following comprehensive experimental approaches:

• Dynamic monitoring of reciprocal regulation:

  • Implement time-course studies using TNS4 antibody-FITC and MET antibodies to track:

    • HGF-induced increases in TNS4 levels (40% increase observed after 30 min)

    • Effects of MET kinase inhibition on TNS4 expression (64% reduction in GTL-16 cells)

    • Temporal relationship between changes in MET and TNS4 expression

  • Apply flow cytometry and immunofluorescence microscopy for quantitative single-cell analysis

  • Develop reporter cell lines with fluorescent-tagged MET to enable dual live-cell imaging

• Molecular mechanism dissection:

  • Study the TNS4-SH2 domain's interaction with phosphorylated MET:

    • Use site-directed mutagenesis to generate the R474A mutation in TNS4

    • Compare wild-type and mutant TNS4 binding to MET using co-immunoprecipitation

    • Assess the impact on downstream signaling pathways (Akt, ERK)

  • Implement proximity ligation assays (PLA) to visualize and quantify TNS4-MET interactions in situ

  • Screen for additional phosphorylation sites on MET that interact with TNS4 beyond Y1313, Y1349, and Y1356

• Trafficking dynamics analysis:

  • Utilize TNS4 antibody-FITC in combination with endocytosis assays to:

    • Track MET internalization rates in cells with varying TNS4 expression

    • Compare basal vs. HGF-induced endocytosis patterns

    • Visualize TNS4 localization during different stages of MET trafficking

  • Apply high-content imaging with automated quantification to analyze:

    • Co-localization of TNS4 and MET in endocytic compartments

    • Recycling rates vs. degradation pathways

    • Impact of TNS4 mutations on MET receptor dynamics

• Signaling pathway integration:

  • Implement multiplexed phospho-flow cytometry using:

    • TNS4 antibody-FITC

    • Phospho-specific antibodies for MET and downstream effectors (p-Akt, p-ERK)

    • Cell cycle markers to correlate with proliferation status

  • Compare signaling outputs across:

    • TNS4-overexpressing cells

    • TNS4-silenced cells

    • Cells expressing TNS4 mutants with impaired MET binding

  • Characterize pathway cross-talk and feedback mechanisms

• Functional impact assessment:

  • Develop cell migration assays with real-time monitoring:

    • Correlate TNS4-FITC signal intensity with migration rates

    • Compare HGF-stimulated vs. basal migration

    • Assess the impact of TNS4 silencing or overexpression

  • Implement 3D invasion models:

    • Spheroid invasion into matrix

    • Organotypic cultures

    • Transwell invasion assays with varying TNS4/MET modulation

• In vivo validation strategies:

  • Generate xenograft models with:

    • Inducible TNS4 expression

    • MET inhibition capabilities

    • Fluorescent reporters for pathway activation

  • Perform intravital imaging using labeled antibodies to track:

    • Tumor growth kinetics

    • Metastatic spread

    • TNS4-MET co-expression patterns in tumor microenvironments

• Translational research applications:

  • Analyze patient-derived samples for TNS4-MET correlation:

    • Use flow cytometry with TNS4 antibody-FITC on fresh tumor samples

    • Correlate expression patterns with clinical outcomes

    • Assess potential as biomarkers for MET-targeted therapies

  • Develop therapeutic targeting strategies:

    • Small molecule inhibitors of TNS4-MET interaction

    • Peptide-based disruptors of the complex

    • Combination approaches with existing MET inhibitors

• Analytical framework for bidirectional regulation:

This comprehensive research strategy leverages TNS4 antibody-FITC to thoroughly investigate the bidirectional regulatory relationship between TNS4 and MET, potentially revealing new therapeutic opportunities for cancers dependent on this signaling axis.

How can TNS4 antibody-FITC be utilized to study the role of TNS4 in T follicular helper cell differentiation and function in vaccine responses?

To investigate TNS4's role in T follicular helper (Tfh) cell differentiation and function in vaccine responses using TNS4 antibody-FITC, researchers can implement this comprehensive experimental framework:

• Baseline expression profiling in T cell subsets:

  • Apply multiparameter flow cytometry combining TNS4 antibody-FITC with markers for:

    • T follicular helper cells (CXCR5, PD-1, Bcl6)

    • T helper subsets (Th1, Th2, Th17, Treg)

    • Memory vs. naïve T cell populations

    • Activation status (CD44, CD69, CD137, OX40)

  • Analyze TNS4 expression across:

    • Secondary lymphoid organs (lymph nodes, spleen)

    • Peripheral blood

    • Tissue-resident memory T cells (particularly in lung)

  • Compare expression patterns before and after vaccination/infection

• Dynamic regulation during Tfh differentiation:

  • Implement in vitro Tfh differentiation systems:

    • Isolate naïve CD4+ T cells

    • Culture under Tfh-polarizing conditions (IL-6, IL-21, low IL-2)

    • Track TNS4 expression throughout differentiation process using TNS4 antibody-FITC

  • Utilize reporter mice or cells:

    • Bcl6 reporters to identify Tfh commitment

    • IL-21 reporters to assess Tfh functionality

    • Correlate with TNS4 expression kinetics

• Functional impact assessment through genetic manipulation:

  • Generate TNS4 knockdown/knockout in primary T cells:

    • CRISPR/Cas9-based approaches

    • shRNA/siRNA strategies

    • Retroviral transduction of dominant-negative constructs

  • Evaluate effects on:

    • Tfh differentiation efficiency

    • Germinal center formation and maintenance

    • B cell help functions

    • Cytokine production (IL-21, IL-4)

    • Migration and positioning within lymphoid tissues

• Integrin-MET-TNS4 complex in Tfh biology:

  • Analyze the TNS4-mediated complex formation in Tfh cells:

    • Co-immunoprecipitation of MET and β1-integrin with TNS4

    • Proximity ligation assays to visualize interactions in situ

    • Assess complex formation during different stages of Tfh differentiation

  • Evaluate functional importance:

    • Implement mutations disrupting specific interactions (e.g., TNS4_R474A)

    • Use specific inhibitors of MET kinase activity

    • Apply integrin-blocking antibodies

• Vaccination/infection models:

  • Apply heterologous infection/immunization strategies as described by Jewell et al. :

    • Prime with intranasal LCMV or intramuscular immunization with adjuvanted recombinant proteins

    • Challenge with recombinant influenza

    • Track TNS4 expression in antigen-specific T cells

  • Compare different vaccine platforms:

    • mRNA vaccines

    • Protein subunit vaccines with various adjuvants

    • Viral vector vaccines

  • Analyze TNS4 expression in relation to:

    • Tfh magnitude and quality

    • Germinal center B cell responses

    • Antibody production (quantity and quality)

    • Memory formation

• Signaling pathway integration:

  • Assess TNS4's role in key Tfh signaling pathways:

    • ICOS-PI3K-Akt axis (given TNS4's role in PI3K/Akt signaling)

    • IL-6/IL-21-STAT3 pathway

    • TCR signal strength and duration

  • Implement phospho-flow cytometry for single-cell signaling analysis:

    • TNS4 antibody-FITC combined with phospho-specific antibodies

    • Time-course analysis after TCR stimulation

    • Comparison between TNS4-high and TNS4-low cells

• TNS4 in Tfh migration and positioning:

  • Analyze TNS4's impact on Tfh localization:

    • Immunofluorescence microscopy of lymphoid tissues

    • Two-photon intravital imaging of Tfh cell migration

    • In vitro migration assays toward CXCL13

  • Assess TNS4-dependent regulation of:

    • Actin cytoskeleton reorganization

    • Integrin activation states

    • Interaction with follicular dendritic cells

• Translational applications:

  • Analyze human vaccination cohorts:

    • Peripheral blood Tfh cells (CXCR5+PD-1+CD4+)

    • TNS4 expression before and after vaccination

    • Correlation with antibody responses

  • Explore TNS4 targeting to enhance vaccine responses:

    • Adjuvants that modulate TNS4 expression

    • Targeted nanoparticles affecting TNS4-integrin interactions

    • Cell-specific delivery systems

• Experimental framework for vaccine studies:

This comprehensive research approach utilizes TNS4 antibody-FITC to elucidate the potentially critical role of TNS4 in Tfh biology and vaccine responses, which could lead to novel strategies for enhancing vaccine efficacy through modulation of TNS4 function.

How can the stability of TNS4-MET-β1-integrin complexes be quantitatively assessed using advanced microscopy techniques with FITC-conjugated antibodies?

To quantitatively assess the stability of TNS4-MET-β1-integrin complexes using advanced microscopy techniques with FITC-conjugated TNS4 antibodies, researchers can implement the following sophisticated methodological approaches:

• Förster Resonance Energy Transfer (FRET) microscopy:

  • Sample preparation:

    • Use TNS4 antibody-FITC as donor and anti-MET or anti-β1-integrin antibodies conjugated with appropriate acceptor fluorophores (e.g., Cy3, TRITC)

    • Optimize antibody concentrations to achieve appropriate donor:acceptor ratios

    • Include appropriate FRET controls (positive and negative)

  • Measurement approaches:

    • Acceptor photobleaching FRET: Measure donor (FITC) intensity before and after acceptor photobleaching

    • Sensitized emission FRET: Measure acceptor emission upon donor excitation with appropriate corrections

    • Fluorescence lifetime imaging microscopy (FLIM)-FRET: Measure changes in FITC fluorescence lifetime due to energy transfer

  • Analysis metrics:

    • Calculate FRET efficiency as quantitative measure of molecular proximity

    • Map spatial distribution of complex formation across cellular structures

    • Correlate FRET efficiency with biological outcomes (e.g., cell migration, proliferation)

• Single-molecule imaging techniques:

  • Total Internal Reflection Fluorescence (TIRF) microscopy:

    • Visualize individual TNS4-MET-β1-integrin complexes at the cell-substrate interface

    • Track complex assembly and disassembly kinetics in real-time

    • Measure dwell times of individual components within complexes

  • Single-molecule tracking:

    • Label TNS4 with low concentrations of FITC-conjugated antibody fragments (Fab)

    • Track diffusion dynamics of individual TNS4 molecules

    • Calculate diffusion coefficients in different cellular compartments

    • Identify transitions between mobile and immobile states (representing complex formation)

  • Super-resolution approaches:

    • Implement single-molecule localization microscopy (PALM/STORM)

    • Achieve 20-30 nm resolution of complex components

    • Perform pair-correlation analysis to quantify co-clustering

    • Map nanoscale organization of complexes in adhesion structures

• Fluorescence Correlation Spectroscopy (FCS) and derivatives:

  • Point FCS:

    • Measure diffusion times of TNS4-FITC in different cellular compartments

    • Detect changes in molecular weight through diffusion coefficient changes

    • Determine concentrations of free vs. complexed TNS4

  • Fluorescence Cross-Correlation Spectroscopy (FCCS):

    • Simultaneously track TNS4-FITC and differently labeled MET or β1-integrin

    • Quantify complex formation through cross-correlation analysis

    • Determine binding affinities and complex stability in living cells

  • Raster Image Correlation Spectroscopy (RICS):

    • Analyze spatial and temporal fluctuations across raster-scanned images

    • Map diffusion and binding parameters throughout the cell

    • Identify regions of stable complex formation

• Fluorescence Recovery After Photobleaching (FRAP) and related techniques:

  • Standard FRAP:

    • Photobleach TNS4-FITC in defined regions

    • Measure recovery kinetics to determine mobile fraction and half-time

    • Compare recovery in adhesion sites vs. other membrane regions

  • Fluorescence Loss In Photobleaching (FLIP):

    • Continuously photobleach one region while monitoring fluorescence in other areas

    • Determine continuity and exchange rates between different TNS4 pools

  • photoactivation/Photoconversion approaches:

    • Use photoactivatable or photoconvertible fluorescent protein-tagged TNS4

    • Track fate of activated molecules to determine retention in complexes

    • Measure dissociation rates under different conditions

• Quantitative colocalization and computational approaches:

  • Advanced colocalization metrics:

    • Implement object-based colocalization analysis

    • Calculate Manders' colocalization coefficients between TNS4, MET, and β1-integrin

    • Use nearest neighbor distance analysis between different components

  • Computational image analysis:

    • Segment adhesion structures using machine learning approaches

    • Quantify relative enrichment of complex components

    • Track temporal evolution of adhesion composition

  • Spatial statistics:

    • Apply Ripley's K-function analysis to quantify clustering

    • Use pair correlation functions to characterize molecular organization

    • Implement tessellation-based approaches to map protein territories

• Perturbation approaches for stability assessment:

  • Pharmacological interventions:

    • Apply MET kinase inhibitors to disrupt phosphorylation-dependent interactions

    • Use cytoskeletal disrupting agents to assess structural requirements

    • Implement PI3K/Akt pathway modulators to assess signaling feedback

  • Force measurements:

    • Combine with Atomic Force Microscopy (AFM) to apply defined forces

    • Assess complex stability under mechanical strain

    • Correlate with cell migration capabilities

  • Temperature and chemical stability:

    • Perform temperature-jump experiments to assess thermodynamic stability

    • Apply increasing detergent concentrations to determine resistance to solubilization

    • Implement crosslinking approaches to capture transient interactions

• Quantitative stability metrics and visualization: