TES Antibody

What is TES protein and why is it significant for molecular research?

TES (Testin) is a 48 kDa focal adhesion protein containing three LIM zinc-binding domains that mediates protein-protein interactions between transcription factors, cytoskeletal proteins, and signaling molecules. TES localizes at cell-cell contacts and along actin stress fibers, interacting with multiple cytoskeletal proteins including Zyxin, Mena, VASP, Talin, and Actin . Its gene maps to chromosome 7q31.2, a common fragile site designated FRA7G, and functions as a potential tumor suppressor . TES is particularly significant in research because its dysregulation is associated with increased cell spreading, decreased motility, and has been implicated in various cancers, making it valuable for studying cellular adhesion mechanisms and tumor biology .

What are the molecular characteristics of available TES antibodies for research?

TES antibodies are available in multiple formats:

Most commercially available antibodies have been validated against human, mouse, and rat TES proteins, with some showing cross-reactivity with other species including bovine, monkey, and chicken samples . The observed molecular weight typically ranges from 38-48 kDa depending on the sample preparation and isoform detected .

What are the optimal dilution ratios for different TES antibody applications?

Based on validated protocols, the following dilution ranges are recommended for TES antibodies:

These parameters should be optimized for specific experimental conditions and antibody lots .

How should researchers design validation experiments for TES antibodies?

According to the International Working Group for Antibody Validation (IWGAV) recommendations, TES antibodies should be validated using at least one of these five approaches :

• Genetic validation: Use knockout/knockdown cells or tissues where TES expression is ablated to confirm antibody specificity.

• Orthogonal validation: Compare antibody-based measurements with non-antibody-based methods (e.g., mass spectrometry or RNA-seq) to verify consistency in TES detection.

• Independent antibody validation: Verify results using multiple antibodies recognizing different epitopes of TES.

• Expression validation: Correlate antibody labeling with expression of tagged TES proteins.

• Immunoprecipitation-mass spectrometry: Confirm TES identity in immunoprecipitated samples using mass spectrometry.

Researchers should document validation methods in publications to ensure reproducibility of results .

How can TES antibodies be utilized to investigate cytoskeletal dynamics?

TES antibodies can be employed in several advanced experimental designs to study cytoskeletal dynamics:

• Co-immunoprecipitation studies: Use TES antibodies to immunoprecipitate TES and associated proteins (Zyxin, Mena, VASP, Talin, and Actin) to map interaction networks under different cellular conditions .

• Live-cell imaging: Combine TES antibody fragments (such as Fabs) with fluorophores for live-cell tracking of TES dynamics at focal adhesions.

• Super-resolution microscopy: Employ TES antibodies with super-resolution techniques (STORM, PALM, STED) to visualize nanoscale organization of TES at cell-cell contacts and along stress fibers.

• Proximity ligation assays (PLA): Detect in situ TES interactions with other cytoskeletal proteins with spatial resolution beyond conventional immunofluorescence.

• Cell migration/adhesion assays: Use TES antibodies to correlate TES localization with cellular behaviors in real-time wound healing or adhesion assays .

What methodological approaches can be used to determine the affinity of TES antibodies?

Determining antibody affinity is crucial for quantitative applications. For TES antibodies, researchers can employ these methodologies:

• Surface Plasmon Resonance (SPR): Measures real-time binding kinetics (kon and koff) to calculate the equilibrium dissociation constant (KD). This approach allows characterization of antibody-antigen interactions without fluorescent labeling .

• Fluorescence ELISA (FL-ELISA): Provides quantitative binding data through fluorescence detection, allowing for determination of KD values with high sensitivity .

• Kinetic Exclusion Assays (KinExA): Measures the concentration of free antibody in solution at equilibrium, enabling accurate KD determination even for high-affinity interactions .

• Bio-Layer Interferometry (BLI): Offers label-free, real-time analysis of antibody-antigen binding kinetics with minimal sample consumption.

The selection of method depends on the expected KD range, sample availability, and required precision. Typical TES antibodies demonstrate KD values in the nanomolar range for their specific epitopes .

What strategies can resolve non-specific binding of TES antibodies?

When encountering non-specific binding with TES antibodies, implement these methodological approaches:

• Optimization of blocking protocols: Test different blocking agents (BSA, non-fat milk, serum) at various concentrations (3-5%) and incubation times (1-2 hours at room temperature).

• Adjustment of antibody concentration: Perform a dilution series to determine the optimal antibody concentration that maximizes specific signal while minimizing background.

• Buffer modification: Increase detergent concentration (0.1-0.3% Triton X-100 or Tween-20) or add low concentrations of SDS (0.01-0.05%) to reduce non-specific interactions.

• Pre-adsorption of antibody: Incubate diluted antibody with cell/tissue lysate from a species different from the target to remove cross-reactive antibodies.

• Alternative fixation methods: For immunocytochemistry/immunohistochemistry, compare different fixatives (PFA, methanol, acetone) as fixation can affect epitope accessibility and non-specific binding.

• Inclusion of appropriate controls: Always include negative controls (isotype control or pre-immune serum) to distinguish between specific and non-specific signals .

How can researchers interpret contradictory results between different TES antibody clones?

When different TES antibody clones yield contradictory results, consider these analytical approaches:

How can TES antibodies contribute to understanding cancer biology?

TES functions as a potential tumor suppressor, and its antibodies can contribute to cancer research through:

• Biomarker development: Quantification of TES expression in tumor vs. normal tissues using validated TES antibodies can help establish TES as a diagnostic or prognostic biomarker.

• Mechanistic studies: Use TES antibodies to investigate the relationship between TES downregulation and tumor progression, particularly in cancers associated with chromosome 7q31.2 alterations .

• Therapeutic target validation: TES antibodies can help validate whether restoring TES function might be a viable therapeutic approach for specific cancer types.

• Pathway analysis: Combine TES antibodies with those against other signaling molecules to map pathways affected by TES dysregulation in cancer cells.

• High-throughput screening: Develop TES antibody-based assays for screening compounds that might restore TES expression or function in cancer cells .

What methodological approaches can improve specificity in computational design of TES-binding antibodies?

Recent advances in computational antibody design can be applied to TES antibodies:

• Biophysics-informed modeling: Computational models can identify distinct binding modes associated with TES epitopes to predict and generate highly specific antibody variants beyond those observed in experimental libraries .

• Multiple-specific selection: Mathematical models can express the probability of an antibody sequence being selected based on selected and unselected modes, enabling design of antibodies with custom specificity profiles .

• High-throughput sequencing analysis: Combining phage display selection with computational analysis allows identification of antibody sequences with either specific high affinity for TES or cross-specificity with related proteins .

• Energy function optimization: By minimizing or maximizing energy functions associated with desired or undesired binding modes, researchers can generate novel TES antibody sequences with predefined binding profiles .

• Experimental validation: Test computationally designed TES antibodies using orthogonal methods to confirm predicted binding properties and specificity .

What are the recommended protocols for immunohistochemical detection of TES?

For optimal immunohistochemical detection of TES:

• Sample preparation:

  • Fix tissues in 10% neutral buffered formalin for 24-48 hours

  • Process and embed in paraffin

  • Section at 4-6 μm thickness

• Antigen retrieval:

  • Primary method: Heat-induced epitope retrieval with TE buffer pH 9.0

  • Alternative: Citrate buffer pH 6.0

  • Heat at 95-98°C for 15-20 minutes

• Blocking and antibody incubation:

  • Block with 3-5% normal serum (matching secondary antibody host) for 1 hour at room temperature

  • Incubate with primary TES antibody (dilution 1:50-1:500) overnight at 4°C

  • Wash 3x with PBS-T

  • Incubate with appropriate HRP-conjugated secondary antibody for 1 hour at room temperature

• Detection and counterstaining:

  • Develop with DAB substrate

  • Counterstain with hematoxylin

  • Dehydrate, clear, and mount with permanent mounting medium

This protocol should be optimized for specific tissue types and antibody batches to ensure optimal staining with minimal background.

How can researchers quantify binding affinity between TES antibodies and their targets?

Accurate quantification of TES antibody binding affinity involves:

• Surface Plasmon Resonance (SPR) protocol:

  • Immobilize purified TES protein on a sensor chip

  • Flow TES antibody at various concentrations (typically 0.1-100 nM)

  • Measure association (kon) and dissociation (koff) rates

  • Calculate KD = koff/kon

  • Include regeneration steps between measurements

  • Perform at least in triplicate for statistical validation

• ELISA-based KD determination:

  • Coat plates with purified TES protein at saturating concentration

  • Incubate with serial dilutions of TES antibody

  • Detect bound antibody with enzyme-conjugated secondary antibody

  • Plot binding curve and fit to appropriate model (typically one-site binding)

  • Extract KD from the resulting curve

  • Include non-linear regression analysis for accurate fitting

• Interpretation of binding parameters:

  • Strong binding: KD < 10 nM

  • Moderate binding: KD = 10-100 nM

  • Weak binding: KD > 100 nM

These quantitative measurements provide critical information for selecting antibodies for specific applications and for comparing different antibody clones .