TTYH2 Antibody, HRP conjugated

What is TTYH2 and why is it significant in research applications?

TTYH2 (Tweety Homolog 2) is a probable large-conductance Ca²⁺-activated chloride channel protein that plays a significant role in Ca²⁺ signal transduction pathways. This protein has emerged as an important research target due to its involvement in cell proliferation and cell aggregation processes . TTYH2 contains five transmembrane domains with an extracellular N-terminus and cytoplasmic C-terminus .

Significantly, TTYH2 has been identified as a potential biomarker in several cancer types, with increased expression associated with cancer progression . Recent research has also identified TTYH2 as a component of volume-regulated anion channels (VRACs) that function independently of the previously characterized LRRC8A-dependent VRACs . This novel role makes TTYH2 antibodies particularly valuable for researchers investigating ion channel biology, cancer progression, and cellular volume regulation mechanisms.

What detection methods can be reliably performed using TTYH2 HRP-conjugated antibodies?

TTYH2 HRP-conjugated antibodies are primarily optimized for Enzyme-Linked Immunosorbent Assay (ELISA) applications . While unconjugated TTYH2 antibodies are often suitable for multiple applications including Western blotting (WB), immunohistochemistry (IHC-p), and immunofluorescence (IF), the HRP-conjugated variants are specifically designed to maximize sensitivity in ELISA-based detection systems .

How does the membrane topology of TTYH2 influence antibody selection and experimental design?

The membrane topology of TTYH2 directly impacts antibody selection strategies for different experimental applications. TTYH2 possesses a complex structure with:

• Five membrane-spanning domains

• Extracellular N-terminus

• Cytoplasmic C-terminus

• Extracellular and cytoplasmic loops containing potential epitopes

This topology creates distinct epitope accessibility challenges depending on the experimental approach:

For instance, the antibody described in search result was generated against three specific TTYH2 peptides: T192 (located in ECL1), T435 and T519 (both located in the CTD). These antibodies were able to specifically detect TTYH2 in transfected cells without cross-reacting with other TTYH family members . When designing experiments, researchers should consider which domain they need to target based on their specific research questions.

What techniques can enhance the sensitivity of TTYH2 HRP-conjugated antibodies in ELISA applications?

Enhancing the sensitivity of TTYH2 HRP-conjugated antibodies involves both optimization of the conjugation process and refinement of detection protocols:

Enhanced Conjugation Through Lyophilization:

Research has demonstrated that incorporating lyophilization into the HRP-antibody conjugation protocol significantly improves sensitivity . This modified conjugation method involves:

• Activation of HRP using sodium metaperiodate (0.15M) to generate aldehyde groups

• Desalting via dialysis with 1× PBS

• Freezing activated HRP at -80°C for 5-6 hours

• Overnight lyophilization of the frozen HRP

• Combining lyophilized HRP with antibody (1:4 molar ratio)

• Stabilization of conjugates via sodium cyanoborohydride

The effectiveness of this method has been demonstrated experimentally, with lyophilized method conjugates showing dramatically improved sensitivity:

This enhancement occurs because "lyophilized activated HRP made it freeze-dried, which reduced reaction volume without changing the amount of both the reactants," effectively increasing the concentration of reactants and improving conjugation efficiency .

How can researchers effectively distinguish between TTYH2 and other TTYH family members in experimental systems?

Distinguishing between TTYH family members (TTYH1, TTYH2, and TTYH3) requires careful consideration of antibody specificity and experimental controls. While these proteins share structural similarities as members of the same family, they exhibit distinct expression patterns and potentially different functions .

Recommended Approach:

• Peptide Selection Strategy: Select antibodies raised against unique peptide sequences that have no homology to other proteins. For example, researchers have successfully developed specific anti-TTYH2 antibodies using peptides spanning residues 192-206 (ECL1), 435-450 (CTD), and 519-534 (CTD) .

• Cross-Reactivity Testing: Before proceeding with experiments, validate antibody specificity by testing against cells expressing each TTYH family member individually. In one study, anti-TTYH2 antibodies were confirmed to "specifically detect TTYH2 in transfected Chinese hamster ovary cells and did not cross-react with lysates from Chinese hamster ovary cells expressing TTYH1-HA or TTYH3-HA" .

• Genetic Validation: Where possible, use genetic approaches (siRNA, CRISPR-Cas9) to validate antibody specificity. For example, researchers targeting TTYH2 with shRNA (target sequence: 5′-GGATTATCTGGACGCTCTTGC-3′) successfully knocked down TTYH2 expression, providing a negative control for antibody specificity testing .

• Domain-Specific Detection: Consider that different domains may be more conserved across family members. C-terminal domains often provide greater specificity for antibody generation compared to transmembrane regions.

What are optimal experimental conditions for investigating TTYH2-protein interactions using immunoprecipitation with HRP-conjugated antibodies?

Investigating TTYH2-protein interactions requires careful optimization of immunoprecipitation (IP) protocols. TTYH2 has been shown to interact with several proteins, including the HECT-type E3 ubiquitin ligase Nedd4-2 and the vesicle transport protein β-COP .

Optimized IP Protocol for TTYH2:

• Cell Lysis Conditions:

  • Use IP lysis buffer containing 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 1 mM EDTA, and 5% glycerol

  • Include protease inhibitor cocktail to prevent degradation

  • Optimize detergent concentration (typically 0.5-1% NP-40 or Triton X-100) to solubilize membrane proteins without disrupting protein-protein interactions

• Antibody Selection:

  • Use antibodies targeting specific domains - research has successfully used antibodies against peptides T192, T435, and T519

  • For detecting interactions with cytoplasmic proteins like Nedd4-2, antibodies targeting the C-terminal domain are particularly effective

• Detection Strategy:

  • For direct detection, use HRP-conjugated TTYH2 antibodies

  • For a sequential approach, perform IP with unconjugated antibodies and detect with HRP-conjugated antibodies in Western blot

• Validation Through Alternative Methods:

  • Confirm interactions using GST pull-down assays - successful protocols have used "50 μg of GST or GST-TTYH2 fusion protein incubated with glutathione-Sepharose 4B beads for 3 h at 4°C"

  • Use reciprocal IPs to verify interactions from both directions

This approach has successfully demonstrated endogenous interaction between TTYH2 and Nedd4-2 in HEK293 cells .

How can TTYH2 HRP-conjugated antibodies be utilized to investigate its role in cancer progression?

TTYH2 has been implicated in cancer progression, with increased expression observed in several cancer types . HRP-conjugated TTYH2 antibodies provide valuable tools for investigating this relationship through multiple experimental approaches:

Cancer Cell Line Profiling:
Researchers can use ELISA with HRP-conjugated TTYH2 antibodies to systematically profile TTYH2 expression across cancer cell line panels. This approach has revealed that TTYH2 is particularly overexpressed in specific cancer types, with functional implications for tumor progression .

Correlation with Clinical Outcomes:
By quantifying TTYH2 expression in patient samples using ELISA and correlating with clinical data, researchers have established that "TTYH2 is correlated with bad prognostic outcomes" . This suggests potential utility as a prognostic biomarker.

Functional Validation through Combined Approaches:
An effective research strategy combines:

• ELISA-based quantification of TTYH2 expression using HRP-conjugated antibodies

• Functional analysis of volume-regulated anion channel (VRAC) currents in cancer cells

• Gene silencing to establish causality

This integrated approach has demonstrated that "VRAC currents were completely absent from TTYH1- and TTYH2-deficient SNU-601 cells, and were clearly restored by expression of TTYH1 or TTYH2" , providing functional validation of TTYH2's role in cancer cell biology.

What considerations are important when investigating post-translational modifications of TTYH2 using antibody-based methods?

TTYH2 undergoes several post-translational modifications (PTMs) that influence its function and localization. Investigating these modifications requires careful consideration of antibody epitopes and detection methods:

N-Glycosylation:
TTYH2 contains N-glycan modifications critical for trafficking to the plasma membrane . When investigating glycosylation:

• Consider that glycosylation may mask epitopes, potentially affecting antibody binding

• Include deglycosylation controls (PNGase F treatment) to assess antibody sensitivity to glycosylation state

• Compare detection in glycosylation-deficient cell lines or following tunicamycin treatment

Ubiquitination:
TTYH2 interacts with the E3 ubiquitin ligase Nedd4-2, suggesting regulation through ubiquitination . Research has shown that:

• TTYH2 contains consensus WW domain binding sites (SP and PY motifs)

• Mutations in these motifs (S444A, S504A, Y509F, and S510A) affect interaction with Nedd4-2

• Detection of ubiquitinated TTYH2 may require immunoprecipitation under denaturing conditions followed by Western blotting

A comprehensive investigation of PTMs should consider both the epitope location relative to modification sites and the potential impact of modifications on protein conformation and antibody accessibility.

What methodological approaches can determine if TTYH2 functions as an independent VRAC or requires association with other proteins?

Recent research has challenged conventional understanding by suggesting TTYH2 can function as a LRRC8A-independent volume-regulated anion channel (VRAC) . Investigating this function requires sophisticated methodological approaches:

Combined Electrophysiology and Immunodetection:
A robust approach involves:

• Patch-clamp electrophysiology to measure VRAC currents under hypotonic conditions

• Concurrent immunodetection of TTYH2 using HRP-conjugated antibodies in ELISA

• Gene silencing or knockout of TTYH2 to establish causality

This approach has demonstrated that "VRAC currents of these cells were abolished by gene silencing of TTYH1 or TTYH2" , supporting a functional role.

Protein Complex Analysis:
To determine if TTYH2 functions independently or requires interaction partners:

• Perform blue native PAGE to identify native TTYH2-containing complexes

• Use co-immunoprecipitation with TTYH2 antibodies followed by mass spectrometry to identify interaction partners

• Use crosslinking approaches to capture transient interactions

Surface Expression Quantification:
The regulation of TTYH2 surface expression is critical to its channel function. Research has shown that "surface expression and activity of TTYH2 were decreased by co-expression with β-COP" , highlighting the importance of trafficking in regulating function.

By integrating these approaches, researchers can more definitively establish whether TTYH2 functions as an independent channel or requires specific protein-protein interactions for its activity.

What strategies can address non-specific binding or high background when using TTYH2 HRP-conjugated antibodies?

Non-specific binding and high background are common challenges when working with HRP-conjugated antibodies. For TTYH2 detection, several specific strategies can improve signal-to-noise ratios:

Optimization of Blocking Conditions:

• Test different blocking agents (BSA, non-fat milk, commercial blocking buffers)

• Optimize blocking time and temperature (typically 1-2 hours at room temperature or overnight at 4°C)

• Consider adding 0.1-0.3% Tween-20 to reduce hydrophobic interactions

Antibody Dilution Optimization:
The manufacturer's guidance that "optimal working dilution should be determined by the investigator" emphasizes the importance of titration experiments. Research indicates that:

• Start with a broad dilution range (1:100 to 1:10,000)

• The enhanced sensitivity from lyophilized conjugation methods may allow dilutions as high as 1:5000

• Compare signal-to-noise ratios rather than absolute signal intensity

Buffer Composition Considerations:
TTYH2 HRP-conjugated antibodies are typically stored in:

• 50% Glycerol

• 0.01M PBS (pH 7.4)

• 0.03% Proclin 300 as preservative

When diluting, maintain buffer components at proportional concentrations to ensure stability, and consider adding stabilizing proteins like 0.1-0.5% BSA to prevent non-specific adsorption to plasticware.

Quality Control Measures:

• Include isotype control antibodies conjugated to HRP

• Perform absorption controls with immunizing peptide

• Use TTYH2-negative and TTYH2-knockdown samples as negative controls

How can researchers validate the functionality and specificity of commercial TTYH2 HRP-conjugated antibodies?

Validating commercial TTYH2 HRP-conjugated antibodies is essential before applying them to critical research questions. A comprehensive validation approach includes:

Expression System Validation:

• Test antibodies on cells with known TTYH2 expression (e.g., LoVo colon cancer cell line)

• Compare with cells where TTYH2 has been knocked down using validated shRNA (target sequence: 5′-GGATTATCTGGACGCTCTTGC-3′)

• Use overexpression systems with tagged TTYH2 constructs as positive controls

Epitope Confirmation:
Most commercial TTYH2 HRP-conjugated antibodies target specific regions of the protein:

• The antibody from search result targets amino acids 455-534

• This C-terminal region is cytoplasmic based on protein topology

Researchers should confirm that the epitope is appropriate for their specific application and accessible under their experimental conditions.

Cross-Reactivity Testing:

• Test against closely related family members (TTYH1, TTYH3)

• Research has confirmed that properly validated antibodies "specifically detect TTYH2 in transfected Chinese hamster ovary cells and did not cross-react with lysates from Chinese hamster ovary cells expressing TTYH1-HA or TTYH3-HA"

Functional Correlation:
For the most rigorous validation, correlate antibody detection with functional measurements:

• Electrophysiological recording of VRAC currents

• Surface expression measurements

• Calcium signaling responses

This multi-faceted approach ensures that the antibody not only detects the correct protein but also provides biologically meaningful information.

How can TTYH2 HRP-conjugated antibodies be incorporated into studies investigating the protein's role in calcium signaling pathways?

TTYH2 is described as a "probable large-conductance Ca²⁺-activated chloride channel" that "may play a role in Ca²⁺ signal transduction" . Investigating this function requires integration of antibody-based detection with functional calcium signaling assays:

Integrated Calcium Imaging and Immunodetection:

• Perform calcium imaging experiments using fluorescent indicators (Fluo-4, Fura-2)

• Fix cells following calcium stimulation protocols

• Detect TTYH2 localization and expression levels using immunocytochemistry

• Correlate TTYH2 expression/localization with calcium transient parameters

Proximity Ligation Assays:
To investigate calcium-dependent protein interactions:

• Use proximity ligation assay (PLA) with TTYH2 antibodies and antibodies against putative interaction partners

• Compare PLA signals under basal conditions and following calcium elevation

• Quantify changes in interaction frequency or intensity

Calcium-Dependent Trafficking Studies:
Research suggests that TTYH2 surface expression is regulated through interaction with trafficking proteins like β-COP . To investigate calcium-dependent trafficking:

• Stimulate cells with calcium ionophores or physiological calcium-elevating stimuli

• Use cell surface biotinylation followed by precipitation and detection with TTYH2 HRP-conjugated antibodies

• Compare surface expression levels before and after calcium elevation

These approaches can help establish the functional relationship between TTYH2 expression, localization, and calcium signaling dynamics in various cell types.

What approaches can determine the structural basis of TTYH2 ion channel function using antibody-based techniques?

Recent cryo-EM studies have revealed that TTYH proteins have "a novel architecture for lipid-interacting proteins" , challenging previous assumptions about their function as ion channels. Antibody-based techniques can complement structural studies to investigate structure-function relationships:

Epitope Mapping for Functional Domains:

• Use a panel of antibodies targeting different TTYH2 domains

• Correlate antibody binding with functional effects (activation, inhibition)

• Identify critical functional epitopes through competitive binding assays

Conformational-Specific Antibody Development:

• Generate antibodies against purified TTYH2 in specific conformational states

• Use these antibodies to track conformational changes during channel activation

• Correlate conformational changes with functional measurements

Structure-Guided Mutagenesis Combined with Antibody Detection:
Based on structural insights:

• TTYH2 contains "a conserved hydrophobic pocket that emerges from the membrane and leads to the extracellular domain"

• This pocket may accommodate "cholesterol or a chemically related compound"

• Create mutations targeting this pocket and assess effects on function

• Use HRP-conjugated antibodies to confirm expression of mutants

Integration with Lipidomic Analysis:
Since TTYH2 may interact with lipids, researchers can:

• Immunoprecipitate TTYH2 from cells

• Extract and analyze co-precipitated lipids by LC-MS

• Correlate lipid binding with functional states

This integrative approach has revealed that "molecules that are enriched in the protein extract compared to the background... could be assigned to phospholipids, sphingolipids, and prostaglandins" , providing insights into potential physiological substrates.

How can researchers utilize TTYH2 HRP-conjugated antibodies in developing novel therapeutic approaches targeting cancer cells?

The discovery that TTYH2 acts as a LRRC8A-independent VRAC in cancer cells suggests potential for novel therapeutic targeting . HRP-conjugated TTYH2 antibodies can facilitate several research approaches in therapeutic development:

High-Throughput Screening Platforms:

• Develop cell-based ELISA assays using HRP-conjugated TTYH2 antibodies

• Screen compound libraries for molecules that modulate TTYH2 expression

• Correlate expression changes with functional effects on cancer cell survival and proliferation

Combinatorial Therapy Assessment:

• Use ELISA to quantify TTYH2 expression in cancer cells treated with standard chemotherapeutic agents

• Identify agents that synergistically modulate TTYH2 expression or function

• Test combination efficacy in cancer models

Biomarker Development:
Since "TTYH2 is correlated with bad prognostic outcomes" , HRP-conjugated antibodies can facilitate:

• Development of quantitative ELISA for TTYH2 in patient samples

• Correlation of TTYH2 levels with treatment response

• Patient stratification for targeted therapies

Therapeutic Antibody Development:
Information from HRP-conjugated antibody binding can inform:

• Epitope selection for therapeutic antibody development

• Screening for antibodies with functional (blocking) activity

• Development of antibody-drug conjugates targeting TTYH2-expressing cells