TTYH2 Antibody, Biotin conjugated

What is TTYH2 and what cellular functions does it perform?

TTYH2 (Protein tweety homolog 2) is a probable large-conductance Ca²⁺-activated chloride channel with five transmembrane domains, featuring an extracellular N-terminus and cytoplasmic C-terminus. It plays critical roles in:

• Ca²⁺ signal transduction pathways

• Cell proliferation processes

• Cell aggregation mechanisms

Research has shown that TTYH2 may be involved in tumor development, with significant upregulation observed in colon cancer tissues compared to normal colonic mucosa (1.230 ± 0.404 vs 0.655 ± 0.373, P = 0.013) . The protein has been implicated in both renal cancer and colon cancer progression . Structurally, TTYH2 consists of 534 amino acids and is encoded by a gene located at chromosomal position 17q24 .

How does biotin conjugation work for TTYH2 antibodies?

Biotin conjugation involves the covalent attachment of biotin molecules to TTYH2 antibodies. This process:

• Creates a high-affinity binding system when combined with streptavidin (Ka ≈ 10¹⁵ M⁻¹)

• Enables sensitive detection through the biotin-streptavidin interaction

• Preserves antibody specificity while adding detection versatility

The biotin-conjugated TTYH2 antibodies typically target specific amino acid sequences within the protein. For example, product ABIN7166230 is designed to bind to amino acids 455-534 of the human TTYH2 protein . The conjugation process uses protein G purification to ensure >95% purity before the biotin attachment .

What are the validated applications for TTYH2 biotin-conjugated antibodies?

TTYH2 biotin-conjugated antibodies have been validated for several experimental applications:

The antibodies have demonstrated reactivity with human TTYH2 and in some cases with mouse and rat homologs . When performing these applications, researchers typically use dilutions between 1:1000-1:5000 for Western blotting, though optimal concentrations should be determined empirically .

How can researchers validate the specificity of TTYH2 biotin-conjugated antibodies?

Validating antibody specificity is critical for reliable results. For TTYH2 biotin-conjugated antibodies, consider these validation approaches:

• Knockdown/Knockout Controls:

  • Utilize TTYH2-specific shRNA to validate specificity (as demonstrated in LoVo cell studies where TTYH2 shRNA significantly reduced both outward and inward chloride currents)

  • Compare signal between knockdown and control samples (e.g., scrambled shRNA)

• Recombinant Protein Controls:

  • Use purified recombinant TTYH2 protein as a positive control

  • Test against recombinant proteins of related family members (TTYH1, TTYH3) to confirm specificity

• Isoform Awareness:

  • Note that at least four isoforms of TTYH2 exist, and some antibodies detect only the two longest isoforms

  • Verify which isoforms your antibody recognizes to properly interpret results

• Cross-Reactivity Testing:

  • Perform parallel experiments in multiple cell lines with known TTYH2 expression profiles

  • Consider testing in colon cancer-derived cell lines like DLD-1, Caco-2, and LoVo, which have demonstrated high TTYH2 expression

How does the TTYH2-β-COP interaction impact experimental design when using TTYH2 antibodies?

The TTYH2-β-COP interaction has significant implications for experimental design:

Binding Mechanism:

• β-COP has been identified as a specific binding partner of TTYH2 through yeast two-hybrid screening

• This interaction occurs specifically at the C-terminal region of TTYH2 (TTYH2-C, amino acids 409-534)

• Co-immunoprecipitation experiments have confirmed this interaction in multiple cell systems

Experimental Considerations:

• Antibodies targeting the C-terminal region of TTYH2 may have reduced accessibility when β-COP is bound

• Surface expression of TTYH2 is decreased by co-expression with β-COP, potentially affecting antibody binding efficiency

• In cells with high β-COP expression, TTYH2 activity and membrane localization are significantly reduced

Methodological Recommendations:

• Include controls measuring β-COP expression when studying TTYH2 in cellular systems

• Consider the subcellular localization of TTYH2 (influenced by β-COP) when interpreting staining patterns

• For functional studies, account for β-COP's inhibitory effect on TTYH2 chloride channel activity

What strategies can minimize biotin interference in immunoassays using TTYH2 biotin-conjugated antibodies?

Biotin interference is a significant concern when using biotin-conjugated antibodies:

Sources of Interference:

• Elevated endogenous biotin from dietary supplements (vitamin B7)

• Biotin-containing components in sample preparation buffers

• Cross-reactivity with other biotinylated proteins in complex samples

Prevention Strategies:

Assay-Specific Considerations:

• In competitive immunoassays, biotin interference typically produces falsely elevated analyte readings

• In sandwich immunoassays, biotin interference tends to cause falsely decreased measurements

• When analyzing immunoassay results, be aware that biotin interference can mimic hyperthyroidism patterns in thyroid function tests

How can proximity labeling techniques be optimized when using TTYH2 biotin-conjugated antibodies?

Proximity labeling with TTYH2 biotin-conjugated antibodies requires specific optimization approaches:

TurboID-Based Proximity Labeling:

• Instead of traditional streptavidin enrichment, consider using biotin-specific antibodies to enrich proximal proteins

• This approach allows identification of biotinylated peptides labeled by TurboID-tagged viral proteins, providing higher confidence in proximity interactions

Methodological Workflow:

• Express TurboID-tagged TTYH2 in the cellular system of interest

• Add biotin to culture medium (typically 50 μM) for labeling (2-24 hours)

• Lyse cells under stringent conditions to minimize non-specific interactions

• Enrich biotinylated proteins using anti-biotin antibodies rather than streptavidin

• Process samples for mass spectrometry analysis

• Apply stringent filtering to identify high-confidence interactors

Critical Parameters:

• Biotin concentration and labeling time significantly impact signal-to-noise ratio

• Cell lysis conditions affect retention of transient interactions

• Wash stringency determines specificity of identified interactions

This approach has proven successful in identifying protein interactions in complex biological systems and can be applied to study TTYH2 interactors in various cellular contexts .

What methodological approaches can elucidate TTYH2's role in cancer progression?

TTYH2 has demonstrated significant associations with cancer progression, particularly in colon and renal cancers. Key methodological approaches include:

Expression Analysis:

• RT-PCR comparison between tumor and adjacent normal tissues (as demonstrated in colon cancer samples showing 1.23 ± 0.404 vs 0.655 ± 0.373 expression levels, P = 0.013)

• Immunohistochemistry with TTYH2 antibodies to visualize protein distribution in tissue sections

Functional Studies:

• siRNA-mediated knockdown of TTYH2 in cancer cell lines (DLD-1, Caco-2, LoVo)

• Evaluation of resulting phenotypes:

  • Cell proliferation (MTT assays)

  • Cell aggregation (aggregation assays)

  • Chloride channel activity (patch-clamp recordings)

Mechanistic Investigations:

• Co-immunoprecipitation studies to identify TTYH2 binding partners

• Whole-cell current recordings to assess TTYH2-mediated chloride currents

• Subcellular localization studies to track TTYH2 trafficking

Data from Established Models:
Studies in colon cancer cell lines revealed that down-regulation of TTYH2 by siRNA resulted in enhanced cell aggregation, suggesting that TTYH2 up-regulation may contribute to reduced cell-cell adhesion and increased metastatic potential .

How do TTYH2 chloride channel functions correlate with tumor phenotypes?

The chloride channel function of TTYH2 has several important correlations with tumor phenotypes:

Electrophysiological Properties:

• TTYH2 mediates calcium-activated, inwardly rectifying anion currents

• In LoVo colon cancer cells, TTYH2 contributes significantly to whole-cell chloride currents:

  • Control cells: -47.75 ± 5.42 pA/pF at -100 mV and +123.28 ± 13.19 pA/pF at +100 mV

  • TTYH2 shRNA-treated cells: -16.77 ± 3.6 pA/pF at -100 mV and +46.22 ± 8.86 pA/pF at +100 mV

Mechanistic Correlations:

• Ion channel activity may regulate:

  • Cell volume regulation during proliferation

  • Calcium signaling pathways influencing gene expression

  • Cell migration through membrane potential modulation

Experimental Approaches:

• Patch-clamp recordings to measure TTYH2-mediated currents in cancer cells

• Calcium imaging to assess the relationship between Ca²⁺ signaling and TTYH2 activity

• Cell migration assays to correlate channel activity with metastatic potential

Recent studies have demonstrated that β-COP overexpression reduces TTYH2 channel activity in LoVo cells, suggesting that trafficking regulation of TTYH2 to the plasma membrane is a critical determinant of its function in cancer cells .

What factors affect the performance of TTYH2 biotin-conjugated antibodies in different applications?

Several factors can impact antibody performance across experimental platforms:

Storage and Handling:

• Recommended storage: Aliquot and store at -20°C

• Avoid repeated freeze-thaw cycles which can lead to degradation

• Some products contain preservatives like 0.03% Proclin-300 which require careful handling

Buffer Composition Effects:

• Typical buffer: 0.01 M PBS, pH 7.4, with 50% glycerol for stability

• Presence of detergents can affect membrane protein epitope accessibility

• Calcium concentrations may alter TTYH2 conformation due to its Ca²⁺-activated nature

Application-Specific Considerations:

Epitope Accessibility:

• TTYH2's five transmembrane domains can limit epitope accessibility

• N-terminal (extracellular) vs. C-terminal (cytoplasmic) targeting antibodies may require different sample preparation techniques

• Consider membrane permeabilization methods when targeting intracellular domains

How can researchers evaluate potential cross-reactivity between TTYH2 antibodies and other tweety family members?

Cross-reactivity assessment is essential for specific TTYH2 detection:

Sequence Homology Analysis:

• TTYH family consists of three members (TTYH1, TTYH2, TTYH3) with significant sequence homology

• Target immunogen sequence (e.g., AA 455-534) should be analyzed for uniqueness within the family

• Antibodies raised against the C-terminal region tend to have higher specificity as this region shows greater sequence divergence

Experimental Validation Approaches:

• Western blot analysis against recombinant TTYH1, TTYH2, and TTYH3 proteins

• Immunocytochemistry in cells transfected with individual family members

• Competitive binding assays using purified TTYH family proteins

Control Systems:

• Use tissues/cells with differential expression of TTYH family members

• Include TTYH2 knockout/knockdown samples as negative controls

• Consider testing in species with known sequence divergence in the target epitope

Some antibodies, like those referenced in result , are designed to detect only specific isoforms (e.g., only the two longest isoforms of TTYH2), which can be advantageous for certain experimental questions but requires careful consideration when interpreting results.

How can TTYH2 biotin-conjugated antibodies be utilized in lipid transport and membrane biology research?

Recent structural studies suggest TTYH2 may have roles beyond chloride channel activity:

Potential Lipid Transport Functions:

• Cryo-EM structures of the TTYH family reveal features suggesting involvement in lipid transport, binding, or metabolism

• Scrambling assays using liposomes containing TTYH2 can assess potential lipid scrambling activity

Experimental Approaches:

• Lipid Scrambling Assays:

  • Reconstitute TTYH2 into liposomes prepared with fluorescent lipid probes (e.g., NBD-PE)

  • Monitor fluorescence changes upon dithionite addition to assess lipid translocation

  • Compare scrambling rates in the presence/absence of calcium (2 mM free calcium vs. calcium-free buffer)

• Membrane Biology Applications:

  • Use biotin-conjugated TTYH2 antibodies to track protein localization during membrane remodeling

  • Combine with lipid probes to investigate co-localization with specific membrane domains

  • Apply super-resolution microscopy techniques for detailed spatial organization analysis

• Protein-Lipid Interaction Studies:

  • Investigate binding of specific lipids to TTYH2 using purified proteins

  • Assess effects of lipid composition on TTYH2 channel activity

The unique architecture of TTYH proteins revealed by structural studies suggests they may represent a novel class of lipid-interacting proteins, opening new research directions beyond their established channel functions .

What are the potential applications of TTYH2 biotin-conjugated antibodies in therapeutic target validation?

As TTYH2 emerges as a potential therapeutic target, biotin-conjugated antibodies offer valuable tools for target validation:

Cancer Therapeutic Applications:

• TTYH2 upregulation in colon and renal cancers suggests potential as a therapeutic target

• Biotin-conjugated antibodies can help validate:

  • Target accessibility in tumor models

  • Expression correlation with disease progression

  • Functional contributions to cancer phenotypes

Targeting Strategy Development:

• EphA2-agonist-biotin-streptavidin complexes have demonstrated effective tumor targeting

• Similar approaches could be developed for TTYH2 targeting using:

  • TTYH2-specific targeting peptides conjugated with biotin

  • Streptavidin-based multivalent complexes for enhanced binding

  • Therapeutic payload delivery systems using the biotin-streptavidin interaction

Validation Methodologies:

• In vitro screening: Using biotin-conjugated antibodies to assess binding to cancer cells vs. normal cells

• In vivo biodistribution: Tracking fluorescently tagged streptavidin-antibody complexes in orthotopic tumor models

• Functional impact assessment: Evaluating effects of TTYH2 targeting on tumor growth and metastasis

These approaches have successfully been applied with EphA2 targeting agents in pancreatic and breast cancer models , providing a template for TTYH2-directed therapeutic development.