TTYH2 Antibody, FITC conjugated

What is TTYH2 and why is it important for research?

TTYH2 is a member of the tweety family of proteins, functioning as a Ca²⁺-activated large conductance chloride (Cl⁻) channel. It contains five transmembrane domains in a 2-2-1 arrangement, also referred to as the Tweety domain . The protein has gained significant research interest due to its up-regulation in colorectal cancer tissues compared to normal colonic mucosa, suggesting its involvement in malignant transformation . TTYH2 appears to play critical roles in tumor cell growth and behavior, potentially influencing cancer cell aggregation and metastatic potential .

What are the optimal storage conditions for TTYH2 Antibody, FITC conjugated?

For maximum stability and antibody performance, TTYH2 Antibody, FITC conjugated should be stored according to the following protocol:

• Upon receipt, aliquot the antibody to minimize freeze-thaw cycles

• Store at -20°C for long-term preservation (stable for up to one year)

• Short-term storage at 4°C is acceptable for up to three months

• Always protect FITC-conjugated antibodies from light exposure to prevent photobleaching

• Avoid repeated freeze-thaw cycles as they significantly reduce antibody activity

The antibody is typically supplied in a buffer containing 0.01 M PBS, pH 7.4, with 0.03% Proclin-300 and 50% glycerol to maintain stability .

What applications is TTYH2 Antibody, FITC conjugated suitable for?

Based on validation data, TTYH2 Antibody, FITC conjugated has been successfully employed in multiple experimental techniques:

Researchers should perform initial titration experiments to determine optimal antibody concentration for their specific experimental setup and sample type .

How can I validate the specificity of TTYH2 Antibody, FITC conjugated in my experimental system?

A comprehensive validation strategy should include:

• Positive and negative control tissues/cells: Use colorectal cancer cell lines (DLD-1, Caco-2, Lovo) as positive controls since they express high levels of TTYH2 . Normal colonic mucosa can serve as a comparative control with lower expression.

• Knockdown/knockout verification: Compare staining in wild-type cells versus those treated with TTYH2-targeting siRNA. Studies have shown successful TTYH2 knockdown in DLD-1 and Caco-2 cell lines .

• Cross-reactivity assessment: Be aware that at least four isoforms of TTYH2 are known to exist, and some antibodies will only detect the two longest isoforms . Verify which isoforms your research requires.

• Peptide blocking: Perform a competitive inhibition experiment using the immunizing peptide (typically a 14 amino acid synthetic peptide near the amino terminus, within amino acids 60-110 of human TTYH2) .

• Multi-technique confirmation: Compare results across different experimental methods (IF, WB, IHC) to ensure consistent detection of the target.

What controls should I include when using TTYH2 Antibody, FITC conjugated for immunofluorescence?

For rigorous immunofluorescence experiments with TTYH2 Antibody, FITC conjugated, incorporate the following controls:

• Primary antibody specificity control: Include a sample incubated with isotype-matched non-specific rabbit IgG-FITC to assess non-specific binding.

• Autofluorescence control: Examine unstained samples to identify any natural fluorescence in your tissue/cells that might interfere with FITC signal interpretation.

• Signal specificity control: Include a peptide competition control where the antibody is pre-incubated with excess immunizing peptide before staining.

• Expression gradient control: Include samples with known differential expression (e.g., normal colon tissue vs. colorectal cancer tissue), as TTYH2 is up-regulated in colorectal cancer (expression ratio of 1.23 ± 0.404 compared to normal tissue) .

• Counterstaining control: Use nuclear counterstains (DAPI/Hoechst) to provide context for cellular localization, as TTYH2 is primarily a transmembrane protein.

What is the expected subcellular localization pattern for TTYH2 and how might this affect my imaging protocol?

TTYH2 is a transmembrane protein with five predicted transmembrane domains , functioning as a Ca²⁺-activated chloride channel. Expect the following localization characteristics:

• Primary localization: Cell membrane, with a distinct membrane staining pattern

• Secondary localization: Potential signals in endoplasmic reticulum and Golgi apparatus during protein processing

• Imaging recommendations:

  • Use confocal microscopy for precise membrane localization

  • Consider z-stack imaging to capture the complete membrane profile

  • Employ membrane-specific counterstains (e.g., wheat germ agglutinin) to confirm co-localization

  • Adjust exposure settings to capture the relatively weak signal often observed with membrane proteins

  • Include super-resolution microscopy for detailed subcellular localization studies if investigating potential protein-protein interactions

How can TTYH2 Antibody, FITC conjugated be utilized to investigate its role in cancer progression?

Based on research showing TTYH2 up-regulation in colorectal cancer and its effects on cell proliferation and aggregation , researchers can employ TTYH2 Antibody, FITC conjugated in several advanced applications:

• Expression correlation studies: Perform multi-label immunofluorescence to correlate TTYH2 expression with cancer progression markers, metastatic potential indicators, and patient survival data.

• Live-cell imaging: Monitor TTYH2 expression and localization during cancer cell migration, invasion, and response to therapeutic agents using the direct FITC conjugation for real-time visualization.

• Protein interaction investigations: Combine with proximity ligation assays to study interactions between TTYH2 and known binding partners such as MANSC1, CCND2, GRB2, and others identified in interactome studies .

• Functional studies: Compare TTYH2 expression before and after manipulation of chloride channel activity to understand structure-function relationships.

• Clinical significance assessment: Create tissue microarrays from patient samples at different disease stages to evaluate TTYH2 as a potential prognostic marker or therapeutic target.

What methodological approaches can resolve contradictory findings regarding TTYH2 function?

When investigating contradictions in the literature regarding TTYH2 function, consider these methodological approaches:

• Isoform-specific analysis: At least four isoforms of TTYH2 are known to exist , and various antibodies may detect different subsets of these isoforms. Design experiments that can distinguish between isoforms using:

  • RT-PCR with isoform-specific primers

  • Western blotting with resolution of different molecular weight bands

  • Recombinant expression of individual isoforms for functional comparison

• Context-dependent functional analysis: TTYH2 may function differently in various cellular contexts. Compare:

  • Normal vs. cancer cells

  • Different cancer types

  • Various stages of cancer progression

  • Responses to different ionic conditions affecting chloride channel function

• Comprehensive signaling analysis: Investigate TTYH2's role in signaling pathways by:

  • Phosphoproteomic analysis following TTYH2 modulation

  • Protein-protein interaction mapping using FITC-labeled TTYH2 antibody in combination with proximity ligation assays

  • Correlation with expression of interacting partners like GRB2, IKBKG, and CCND2

How can chloride channel functionality of TTYH2 be correlated with its potential role in cancer using FITC-conjugated antibodies?

To investigate the relationship between TTYH2's chloride channel function and its cancer-related roles using FITC-conjugated antibodies:

• Combined electrophysiology and imaging:

  • Perform patch-clamp analysis of chloride currents while simultaneously imaging TTYH2-FITC localization

  • Correlate channel activity with expression levels and subcellular distribution

• Calcium-dependency studies:

  • Use calcium ionophores or chelators to modulate intracellular calcium while monitoring TTYH2 localization with the FITC-conjugated antibody

  • Quantify translocation or clustering upon calcium activation

• Functional mutation analysis:

  • Compare wild-type TTYH2 expression (using FITC-labeled antibody) with expression of chloride-inactive mutants

  • Evaluate differences in cancer-related phenotypes (proliferation, aggregation, migration)

• Microenvironmental manipulation:

  • Alter extracellular chloride concentrations while monitoring TTYH2 expression and localization

  • Correlate changes with metastatic behaviors in cancer cell models

• Therapeutic target validation:

  • Use FITC-conjugated TTYH2 antibodies to monitor responses to chloride channel modulators

  • Quantify both channel function and cancer-related phenotypes to establish causality

What are the common causes of weak or non-specific signal when using TTYH2 Antibody, FITC conjugated?

When troubleshooting suboptimal results with TTYH2 Antibody, FITC conjugated, consider these common issues and solutions:

How can I optimize TTYH2 Antibody, FITC conjugated for use in flow cytometry?

While not explicitly listed in all product specifications, FITC-conjugated antibodies are generally suitable for flow cytometry. Follow these optimization steps:

• Initial titration: Test a range of antibody concentrations (typically 0.1-10 μg/ml) to determine the optimal signal-to-noise ratio.

• Cell preparation considerations:

  • For intracellular/transmembrane domains: Use appropriate permeabilization reagents (0.1% saponin or commercial permeabilization buffers)

  • For extracellular domains: Avoid harsh permeabilization to preserve epitope integrity

• Controls setup:

  • Include unstained cells for autofluorescence assessment

  • Use isotype control (rabbit IgG-FITC) at the same concentration

  • Include a known positive sample (e.g., DLD-1 or Caco-2 cells)

  • Consider a blocking peptide control

• Signal optimization:

  • Adjust PMT voltage for optimal FITC detection

  • Implement compensation if using multiple fluorophores

  • Consider fixation only after staining if epitope is sensitive to fixatives

• Analysis strategy:

  • For heterogeneous populations, use co-staining with cell-type specific markers

  • Correlate TTYH2 expression with functional parameters or other markers of interest

How do I effectively design experiments to study TTYH2 interactions with other proteins using FITC-conjugated antibodies?

To investigate TTYH2's interactions with its numerous potential binding partners , consider these experimental approaches using FITC-conjugated antibodies:

• Co-localization studies:

  • Perform dual immunofluorescence with TTYH2 Antibody-FITC and antibodies against potential interaction partners (e.g., MANSC1, GRB2, CCND2) labeled with spectrally distinct fluorophores

  • Utilize high-resolution confocal microscopy with colocalization analysis (Pearson's correlation, Manders' overlap coefficient)

  • Include appropriate controls for bleed-through and non-specific binding

• Proximity-based interaction assays:

  • Implement Förster Resonance Energy Transfer (FRET) between FITC-labeled TTYH2 and acceptor fluorophore-labeled potential partners

  • Consider Proximity Ligation Assay (PLA) to visualize protein interactions within 40nm distance

  • Use BiFC (Bimolecular Fluorescence Complementation) for direct interaction visualization

• Dynamic interaction studies:

  • Apply Fluorescence Recovery After Photobleaching (FRAP) to assess mobility changes upon potential partner binding

  • Implement live-cell imaging with FITC-labeled antibody fragments to monitor dynamic interactions

  • Correlate interaction patterns with functional changes in chloride channel activity

• Quantitative interaction analysis:

  • Combine imaging with co-immunoprecipitation to confirm interactions

  • Validate interactions in multiple cell types, including cancer and normal cells

  • Investigate how interactions change under different conditions (calcium levels, cell cycle stages, differentiation states)

What emerging technologies could enhance TTYH2 research using fluorescently labeled antibodies?

Several cutting-edge technologies could significantly advance TTYH2 research when combined with fluorescently labeled antibodies:

• Super-resolution microscopy (STORM, PALM, STED): These techniques overcome the diffraction limit, allowing visualization of TTYH2 distribution within membrane microdomains and precise localization relative to interaction partners at nanometer resolution.

• Single-molecule tracking: Using photostable fluorophore-conjugated antibody fragments to track individual TTYH2 molecules in living cells, providing insights into dynamic behavior, diffusion patterns, and clustering.

• Mass cytometry (CyTOF) with fluorescent antibody reporters: Combining mass cytometry with fluorescent antibodies allows comprehensive phenotyping of TTYH2-expressing cells across multiple parameters simultaneously.

• Spatial transcriptomics with protein detection: Correlating TTYH2 protein expression (via FITC-antibody) with localized transcriptome analysis to understand gene expression networks in TTYH2-rich regions.

• Organoid and patient-derived xenograft (PDX) models: Implementing advanced 3D culture systems with multiplexed imaging to study TTYH2 in more physiologically relevant contexts.

How might TTYH2 antibodies contribute to understanding the relationship between chloride channel function and cancer biology?

TTYH2 antibodies, including FITC-conjugated variants, can bridge fundamental ion channel research with cancer biology through:

• Simultaneous functional-structural studies: Combining electrophysiological recordings with real-time antibody-based imaging to correlate channel activity with protein localization and complex formation.

• Tumor microenvironment analysis: Using FITC-conjugated TTYH2 antibodies to investigate how ionic microenvironment changes affect TTYH2 expression and function in tumor vs. normal tissues.

• Therapeutic target validation: Employing antibodies to monitor TTYH2 expression changes in response to interventions targeting chloride channel function, providing direct evidence for mechanistic connections.

• Cancer subtype classification: Developing TTYH2 expression profiles across cancer types and stages to identify patterns that correlate with specific dependencies on chloride channel activity.

• Resistance mechanism elucidation: Investigating whether alterations in TTYH2 expression or localization contribute to therapy resistance through ion transport-dependent survival mechanisms.

What experimental approaches could clarify the significance of TTYH2 upregulation in cancer progression?

To determine whether TTYH2 upregulation is causally linked to cancer progression or merely a consequence, consider these experimental approaches:

• Temporal expression analysis:

  • Use FITC-conjugated TTYH2 antibodies to track expression changes during cancer initiation, progression, and metastasis in controlled model systems

  • Correlate expression timing with acquisition of malignant phenotypes

• Gain/loss-of-function studies with molecular monitoring:

  • Create inducible TTYH2 expression or knockdown systems

  • Monitor phenotypic changes using multiparameter imaging with FITC-conjugated antibodies

  • Compare effects of wild-type vs. channel-inactive mutants

• Patient-derived models with longitudinal analysis:

  • Establish patient-derived organoids or xenografts

  • Track TTYH2 expression during treatment response and resistance development

  • Correlate with clinical outcomes and treatment response

• Mechanistic pathway dissection:

  • Identify signaling cascades activated by TTYH2 upregulation

  • Determine whether these pathways are essential for maintaining malignant phenotypes

  • Use combination approaches targeting both TTYH2 and downstream effectors

• Meta-analysis of clinical samples:

  • Develop standardized TTYH2 immunostaining protocols

  • Create a scoring system based on expression level, subcellular distribution, and correlation with prognostic markers

  • Validate across multiple cancer types and clinical outcomes