TNS4 Antibody, Biotin conjugated

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

TNS4 (also known as cten) is a protein that directly interacts with phosphorylated MET tyrosine kinase receptor via its SH2 domain to positively regulate cell survival, proliferation, and migration through increased MET protein stability. Unlike other tensins, TNS4 lacks an actin-binding domain and has been identified as a putative oncogene in many cancer types . TNS4 is significantly upregulated in colorectal, lung, ovarian, and gastric cancers, making it an important target for cancer research . Additionally, TNS4 plays a critical role in tubulogenesis and epithelial sheet invasion, highlighting its importance in developmental biology studies .

What are the structural characteristics of TNS4 that make antibody generation feasible?

TNS4 contains several functional domains that serve as useful epitope targets for antibody generation:

• SH2 domain: Mediates binding to phosphorylated MET receptor (with R474 as a critical residue)

• PTB domain: Facilitates interaction with β1-integrin

• Lacks the actin-binding domain found in other tensins like TNS3

These distinctive structural features allow for generation of specific antibodies that can recognize TNS4 without cross-reactivity to other tensin family members.

How does biotin conjugation enhance the utility of TNS4 antibodies in research?

Biotin conjugation significantly enhances TNS4 antibody utility through several mechanisms:

• Signal amplification: An antibody conjugated with several biotin molecules can amplify signal through the tetravalent binding of streptavidin/avidin, thereby increasing sensitivity in various assays

• Versatile detection options: Biotinylated antibodies can be visualized using streptavidin or avidin conjugated to various reporters (enzymes, fluorophores, etc.)

• Stable conjugation: The bond formed between biotin and streptavidin/avidin is exceptionally strong, ensuring reliable detection

• Compatibility with multiple techniques: Biotinylated TNS4 antibodies can be used in Western blotting, IHC, ELISA, and flow cytometry with appropriate streptavidin detection systems

What is the optimal protocol for biotinylating an anti-TNS4 antibody while preserving its binding activity?

For optimal biotinylation of anti-TNS4 antibodies while preserving activity:

• Buffer preparation: Use a protein-compatible buffer (e.g., 0.02M potassium phosphate, 0.15M sodium chloride, pH 7.2) that is free of primary amines

• Reagent preparation: Immediately before use, prepare 20mM Biotin-PEG4-NHS Ester in DMSO or DMF

• Conjugation reaction:

  • For dilute antibody solutions (1 mg/ml): Use ≥20-30-fold molar excess of biotin

  • For concentrated antibody solutions (5-10 mg/ml): Use ≥10-fold molar excess of biotin

• Incubation conditions: Either:

  • Room temperature for 30 minutes, or

  • On ice for 2 hours

• Purification: Remove non-reactive reagent by dialysis or desalting

• Storage: Store in 0.02M potassium phosphate, 0.15M sodium chloride, pH 7.2 with 0.01% sodium azide and 10 mg/mL BSA (immunoglobulin and protease-free) for optimal stability

How can I verify the degree of biotinylation of my TNS4 antibody and what is the optimal biotin:antibody ratio?

To verify biotinylation and determine the optimal ratio:

• Quantification methods:

  • HABA assay: Colorimetric quantification based on displacement of HABA dye from avidin

  • FluoReporter™ assay: Fluorescence-based determination of biotin incorporation

  • UV-Vis spectroscopy: Non-destructive quantification if using biotinylation reagents with built-in signal capabilities

• Optimal ratios:

  • For most applications, 3-8 biotin molecules per antibody is optimal

  • Over-biotinylation (>10 biotins per antibody) may lead to:

    • Loss of antibody activity

    • Increased non-specific binding

    • Potential aggregation

• Functional validation:

  • Test antibody binding pre- and post-biotinylation using ELISA against recombinant TNS4

  • Compare immunoprecipitation efficiency of native and biotinylated antibody

What controls should be included when using biotinylated TNS4 antibodies in experimental systems?

For rigorous experimental design, include these controls:

• Specificity controls:

  • Isotype-matched biotinylated control antibody (unrelated specificity)

  • Pre-absorption of biotinylated TNS4 antibody with recombinant TNS4 protein

  • TNS4 knockdown or knockout samples (negative control)

  • Recombinant TNS4-expressing cells (positive control)

• Biotin-specific controls:

  • Non-biotinylated primary antibody with same detection system

  • Detection reagent (streptavidin-conjugate) alone to assess endogenous biotin

  • Biotin blocking step if working with tissues containing high endogenous biotin

• Method-specific controls:

  • For Western blot: Molecular weight markers to confirm specificity (TNS4 is ~77 kDa)

  • For IHC/ICC: Secondary antibody alone controls

  • For FACS: Fluorescence minus one (FMO) controls

How can I quantify TNS4 protein levels using biotinylated antibodies in different assay formats?

Western Blot Quantification:

• Perform standard western blotting using biotinylated TNS4 antibody

• Detect with streptavidin-HRP or streptavidin-AP conjugate

• Capture images with a digital imaging system

• Quantify band intensity using software (ImageJ, etc.)

• Normalize TNS4 signals to loading controls (β-actin, GAPDH)

• Compare relative expression across samples using integrated densities

ELISA Quantification:

• Generate a standard curve using recombinant TNS4 protein

• Measure samples in technical triplicates

• Calculate TNS4 concentration based on the standard curve

• Validate with spike-recovery experiments

Flow Cytometry Quantification:

• Use biotinylated TNS4 antibody followed by fluorochrome-conjugated streptavidin

• Analyze median fluorescence intensity (MFI)

• Calculate fold-change relative to control samples

• Use quantitative beads to determine absolute molecule counts if needed

How can I differentiate between TNS4 and other tensin family members (TNS1, TNS2, TNS3) in my experiments?

Differentiating TNS4 from other tensin family members requires careful experimental design:

• Antibody selection strategy:

  • Target unique regions of TNS4 that lack homology with other tensins

  • The absence of the actin-binding domain in TNS4 (present in TNS1-3) provides a distinguishing feature

  • Select antibodies targeting the C-terminal region containing the SH2 and PTB domains

• Validation approaches:

  • Western blotting with recombinant tensins to confirm specificity

  • Size discrimination (TNS4 is smaller than other tensins)

  • Use cells with known expression patterns of different tensins as positive/negative controls

• Molecular techniques for validation:

  • siRNA knockdown of TNS4 specifically (verify with qRT-PCR)

  • Expression of RNAi-resistant TNS4 constructs for rescue experiments

  • Analysis of SH2-domain dependence using the R474A mutation specific to TNS4

What are the expected TNS4 expression patterns in different cell types and how do they correlate with MET and β1-integrin expression?

Based on the research literature, TNS4 expression patterns and correlations include:

Expression Patterns:

• Cancer cells: Significantly upregulated in colorectal, lung, ovarian, and gastric cancers

• Normal tissues: Generally lower expression compared to matched tumors

• Cell lines: High expression in cancers with MET amplification (e.g., GTL-16 gastric carcinoma cells)

Correlation with MET and β1-integrin:

• TNS4 forms a complex with both MET and β1-integrin in cell adhesion sites

• TNS4 positively regulates both MET and β1-integrin stability:

  • TNS4 silencing reduces MET levels by 28-59%

  • TNS4 silencing reduces β1-integrin levels by 26-55%

• TNS4-MET binding occurs via the SH2 domain of TNS4 and is dependent on MET phosphorylation

• TNS4-β1-integrin binding occurs via the PTB domain and is not dependent on growth factor stimulation

Relevant subcellular localization patterns:

• TNS4, MET, and β1-integrin colocalize in paxillin-positive adhesion sites

• Upon HGF stimulation, colocalization of TNS4 and MET significantly increases

What are common issues when using biotinylated TNS4 antibodies and how can they be resolved?

High Background Signal:

• Cause: Excessive biotinylation, endogenous biotin, or non-specific binding

• Solutions:

  • Use antibodies with optimal biotin:antibody ratio (3-8 biotins per antibody)

  • Add avidin/streptavidin blocking step before primary antibody incubation

  • Increase BSA concentration in blocking buffer (up to 5%)

  • Use biotin-free BSA for blocking and antibody dilution

Weak or No Signal:

• Cause: Insufficient biotinylation, degraded antibody, or low target expression

• Solutions:

  • Verify biotinylation using HABA or FluoReporter™ assays

  • Optimize antibody concentration

  • Extend incubation time with streptavidin conjugate

  • Use signal amplification methods like ABC (Avidin-Biotin Complex)

Non-specific Bands in Western Blot:

• Cause: Cross-reactivity or degradation of target protein

• Solutions:

  • Include protease inhibitors during sample preparation

  • Verify antibody specificity using TNS4 knockdown cells

  • Optimize antibody concentration and washing conditions

  • Pre-absorb antibody with recombinant TNS1-3 proteins

How can I troubleshoot issues with TNS4 detection in different subcellular compartments?

TNS4 localizes to different subcellular compartments depending on cellular context. Troubleshooting compartment-specific detection issues:

Membrane Localization Issues:

• Problem: Weak membrane staining despite expected membrane localization

• Solutions:

  • Use gentle fixation (2-4% PFA for 10-15 minutes)

  • Avoid harsh permeabilization (use 0.1% Triton X-100 or 0.05% saponin)

  • Stain for β1-integrin as co-localization marker

  • Use the "Staining Membrane-associated Proteins protocol" as referenced in

Adhesion Site Detection:

• Problem: Difficulty visualizing TNS4 at adhesion sites

• Solutions:

  • Co-stain with paxillin as adhesion site marker

  • Use cells plated on fibronectin or collagen to enhance adhesion site formation

  • Perform HGF stimulation (10-50 ng/ml for 15-30 minutes) to enhance TNS4 recruitment

  • Use super-resolution microscopy techniques for better resolution of adhesion structures

Nuclear Detection:

• Problem: Unexpected nuclear staining

• Solutions:

  • Verify specificity with knockdown controls

  • Check for potential nuclear localization signals in TNS4 sequence

  • Perform fractionation experiments to biochemically validate localization

What are the critical factors that affect the stability of biotinylated TNS4 antibodies and how can longevity be maximized?

To maximize stability of biotinylated TNS4 antibodies:

Storage Conditions:

• Store lyophilized antibody at -20°C to -70°C for up to 12 months

• After reconstitution, store at 2-8°C for up to 1 month under sterile conditions

• For long-term storage after reconstitution, aliquot and store at -20°C to -70°C for up to 6 months

• Avoid repeated freeze-thaw cycles (no more than 3 cycles)

Buffer Composition:

• Optimal buffer: 0.02M potassium phosphate, 0.15M sodium chloride, pH 7.2

• Include stabilizers: 10 mg/mL BSA (immunoglobulin and protease-free)

• Add preservative: 0.01% (w/v) sodium azide

• Avoid buffers containing primary amines (Tris, glycine) which can compete with biotinylation sites

Handling Recommendations:

• Reconstitute with deionized water or equivalent

• Centrifuge briefly after thawing to collect all material

• Keep on ice during experimental procedures

• Avoid exposure to strong light (particularly for dual-labeled antibodies)

• Use low protein-binding tubes for storage

How can biotinylated TNS4 antibodies be used to study the TNS4-MET-β1-integrin axis in cancer progression models?

Advanced research applications for studying this important signaling axis include:

Multi-protein Complex Analysis:

• Sequential Immunoprecipitation:

  • First IP: Capture with biotinylated TNS4 antibody on streptavidin beads

  • Elution with biotin

  • Second IP: With MET or β1-integrin antibodies

  • Analysis demonstrates proteins in the same complex

• Proximity Ligation Assay (PLA):

  • Use biotinylated TNS4 antibody with streptavidin-conjugated oligonucleotides

  • Pair with antibodies against MET or β1-integrin

  • Quantify interactions through fluorescent signal representing <40nm proximity

Functional Studies in Cancer Models:

• Xenograft Tumor Models:

  • Establish cancer cell lines with modulated TNS4 expression

  • Inject subcutaneously in mice (2×10^6 cells)

  • Monitor tumor growth for 12 days

  • Analyze tissues using biotinylated TNS4 antibody with Ki67 and TUNEL staining

• Real-time Invasion Monitoring:

  • Implement time-lapse microscopy to track TNS4-expressing cells during invasion

  • Use biotinylated TNS4 antibody for immunofluorescence at fixed timepoints

  • Correlate TNS4 levels with invasive capacity

• Domain-specific Function Analysis:

  • Employ TNS4 mutants (e.g., R474A SH2 domain mutant)

  • Compare wild-type and mutant effects on MET stability and trafficking

  • Use biotinylated antibodies for detection in biochemical and imaging assays

What are the latest methodologies for studying TNS4-dependent MET trafficking using biotinylated antibodies?

Cutting-edge approaches for studying TNS4-mediated MET trafficking include:

Cell Surface Biotinylation-Based Endocytosis Assay:

• Biotinylate cell surface proteins with non-membrane permeable biotin reagents

• Allow endocytosis to proceed at specific timepoints

• Remove remaining surface biotin with reducing agents

• Immunoprecipitate MET and detect internalized fraction with streptavidin-HRP

• Quantify endocytosis rates between control and TNS4-manipulated cells

Antibody-Based Trafficking Assays:

• FACS-Based Endocytosis Measurement:

  • Label surface MET with antibodies at 4°C

  • Allow internalization at 37°C for varying times

  • Detect remaining surface MET by flow cytometry

  • Compare cells expressing wild-type TNS4 versus TNS4_R474A mutant

• Live-Cell Imaging with Quantum Dots:

  • Label biotinylated TNS4 antibodies with streptavidin-conjugated quantum dots

  • Perform real-time confocal microscopy to track TNS4 movement

  • Co-label MET using spectrally distinct fluorophores

  • Analyze trafficking dynamics under HGF stimulation

Advanced Analytical Approaches:

• Super-Resolution Microscopy:

  • Use dSTORM or PALM techniques with biotinylated antibodies

  • Achieve 20-50nm resolution of TNS4-MET-integrin complexes

  • Analyze nanoscale organization in adhesion structures

• Correlative Light and Electron Microscopy (CLEM):

  • Detect TNS4 with biotinylated antibodies and streptavidin-gold particles

  • Combine fluorescence and electron microscopy data

  • Characterize ultrastructural features of TNS4-positive endocytic vesicles

How can multi-parametric analysis be performed using biotinylated TNS4 antibodies in combination with other cancer biomarkers?

For sophisticated multi-parametric analyses of TNS4 and other cancer biomarkers:

Multiplexed Immunohistochemistry:

• Sequential Multiplexing Protocol:

  • Stain with biotinylated TNS4 antibody and streptavidin-HRP

  • Develop with spectrally distinct chromogen (e.g., DAB)

  • Strip or quench

  • Repeat with additional biomarkers (MET, β1-integrin, proliferation markers)

  • Use multispectral imaging for analysis

• Tyramide Signal Amplification (TSA) Multiplexing:

  • Use biotinylated TNS4 antibody with streptavidin-HRP

  • Amplify signal with tyramide-fluorophore conjugates

  • Perform heat-mediated antibody stripping

  • Repeat for up to 7-8 additional markers

  • Analyze using multispectral imaging systems

Advanced Flow Cytometry Applications:

• Mass Cytometry (CyTOF):

  • Conjugate anti-TNS4 antibodies with distinctive metal isotopes

  • Combine with antibodies against other cancer markers

  • Analyze >40 parameters simultaneously at single-cell resolution

  • Identify rare subpopulations with distinctive TNS4/MET/integrin profiles

• Spectral Flow Cytometry:

  • Use biotinylated TNS4 antibody with streptavidin-fluorophore conjugates

  • Combine with 12-15 additional fluorescently labeled antibodies

  • Employ spectral unmixing algorithms for analysis

  • Correlate TNS4 expression with other cancer stem cell markers

Computational Analysis Approaches:

• Apply machine learning algorithms to identify patterns in multi-parameter datasets

• Perform dimensionality reduction (tSNE, UMAP) to visualize complex relationships

• Create predictive models correlating TNS4 expression patterns with patient outcomes

• Integrate with genomic and transcriptomic data for comprehensive cancer profiling

What are emerging technologies that might enhance detection and analysis of TNS4 using biotinylated antibodies?

Several emerging technologies hold promise for advancing TNS4 research:

• CRISPR-based Tagging:

  • CRISPR knock-in of biotin acceptor peptides to endogenous TNS4

  • Expression of BirA ligase for in-cell biotinylation

  • Detection with streptavidin conjugates without antibodies

• Single-Molecule Detection Methods:

  • Single-molecule pull-down (SiMPull) assays using biotinylated antibodies

  • Direct visualization of individual TNS4-containing complexes

  • Quantification of complex stoichiometry and heterogeneity

• Spatial Transcriptomics Integration:

  • Combine biotinylated TNS4 antibody detection with spatial transcriptomics

  • Correlate protein localization with gene expression patterns

  • Map TNS4 protein-RNA relationships in tissue context

• Organoid and Patient-Derived Xenograft Applications:

  • Apply biotinylated TNS4 antibodies for 3D organoid imaging

  • Develop clearing techniques compatible with streptavidin detection systems

  • Create high-content screening platforms for TNS4-targeted therapeutics

What is the current understanding of TNS4's role in treatment resistance, and how might biotinylated antibodies help address this challenge?

TNS4's emerging role in treatment resistance and research approaches:

Current Understanding:

• TNS4 stabilizes MET, potentially contributing to resistance against MET-targeted therapies

• TNS4-dependent MET trafficking regulation may alter drug accessibility to targets

• TNS4 expression correlates with increased survival of MET-dependent tumors

Research Approaches Using Biotinylated Antibodies:

• Patient Sample Analysis:

  • Multiplex IHC with biotinylated TNS4 antibodies on pre/post-treatment samples

  • Correlate TNS4 levels with treatment response

  • Identify threshold levels predictive of resistance

• Therapeutic Monitoring:

  • Track changes in TNS4-MET-β1-integrin complex formation during treatment

  • Use biotinylated antibodies for immunoprecipitation from patient biopsies

  • Develop companion diagnostic approaches

• Combination Therapy Rationale:

  • Screen for compounds that disrupt TNS4-MET interaction using competitive binding assays

  • Utilize biotinylated TNS4 antibodies to monitor complex disruption

  • Identify synergistic combinations that overcome TNS4-mediated resistance