TBC1D4 Antibody, Biotin conjugated

What is TBC1D4 and why is it significant in metabolic research?

TBC1D4, also known as AS160 (Akt Substrate of 160 kDa), is a Rab GTPase-activating protein (RabGAP) that plays a critical role in insulin-regulated glucose uptake. The protein modulates the translocation of glucose transporter 4 (GLUT4) to the plasma membrane in skeletal muscle and adipose tissue. Its significance lies in its position as a nexus for insulin- and contraction-responsive signals in metabolic regulation, potentially mediating enhanced insulin action in skeletal muscle after exercise . TBC1D4 has emerged as an important research target for understanding insulin resistance and type 2 diabetes, as impaired phosphorylation of TBC1D4 at sites like Thr-642 correlates with defective GLUT4 translocation in diabetic conditions.

What are the technical specifications of commercially available TBC1D4 Antibody, Biotin conjugated?

The TBC1D4 Antibody, Biotin conjugated is a polyclonal antibody derived from rabbit hosts, targeted against recombinant human TBC1D4 protein (amino acids 1-94) . The antibody has the following specifications:

The biotin conjugation enhances detection sensitivity and versatility in various assay formats .

How should TBC1D4 Antibody, Biotin conjugated be stored to maintain optimal activity?

For optimal preservation of antibody activity, TBC1D4 Antibody, Biotin conjugated should be stored at -20°C or -80°C upon receipt . The antibody should be aliquoted before freezing to minimize freeze-thaw cycles, which can significantly reduce antibody effectiveness . When handling the antibody, it's crucial to avoid exposure to light due to the light-sensitive nature of the biotin conjugate . The buffer containing 50% glycerol helps maintain stability during freezing . When working with the antibody, thaw aliquots on ice and return unused portions to -20°C immediately after use. Under these storage conditions, the antibody typically maintains activity for at least 12 months, though specific shelf-life may vary between manufacturers .

What are the optimal sample preparation methods for detecting TBC1D4 in human skeletal muscle?

For effective detection of TBC1D4 in human skeletal muscle samples, researchers should follow these methodological steps:

• Tissue Collection and Preservation: Muscle biopsies should be snap-frozen in liquid nitrogen immediately after collection and stored at -80°C until analysis to preserve phosphorylation status .

• Homogenization: Frozen muscle samples (approximately 20-30 mg) should be pulverized and homogenized in ice-cold buffer containing phosphatase inhibitors (sodium fluoride, sodium pyrophosphate, sodium orthovanadate) and protease inhibitors to prevent protein degradation and preserve phosphorylation states .

• Protein Extraction and Quantification: After homogenization, samples should be centrifuged (typically 10,000-15,000 g for 10-20 minutes at 4°C) to remove insoluble material. The supernatant should be collected and protein concentration determined using standard methods like Bradford or BCA assay .

• Denaturation: Prior to SDS-PAGE, samples should be denatured in Laemmli buffer containing SDS and a reducing agent like β-mercaptoethanol, heated at 95°C for 5 minutes .

This preparation ensures preservation of TBC1D4 and its phosphorylation status for subsequent detection with the biotin-conjugated antibody in various applications .

What dilution ranges are recommended for different applications of TBC1D4 Antibody, Biotin conjugated?

The optimal dilution of TBC1D4 Antibody, Biotin conjugated varies depending on the specific application:

While these ranges provide a starting point, researchers should note that optimal dilutions/concentrations should be determined experimentally by the end user for each specific application and detection system . Factors that affect optimal dilution include sample type, protein abundance, detection method sensitivity, and experimental conditions. A titration experiment is recommended when first establishing protocols with this antibody .

How can researchers validate the specificity of TBC1D4 Antibody, Biotin conjugated?

To validate the specificity of TBC1D4 Antibody, Biotin conjugated, researchers should employ several complementary approaches:

• Positive and Negative Controls: Include lysates from tissues known to express high levels of TBC1D4 (e.g., skeletal muscle, adipose tissue) as positive controls and tissues with minimal expression as negative controls .

• Blocking Peptide Assays: Pre-incubate the antibody with excess immunizing peptide (recombinant TBC1D4 protein fragment 1-94AA) before application to samples. Specific binding should be significantly reduced or eliminated .

• siRNA/shRNA Knockdown: For cell culture experiments, compare detection between control cells and cells where TBC1D4 expression has been knocked down using RNA interference methods. Specific binding should show reduced signal in knockdown samples .

• Molecular Weight Verification: Confirm that the detected protein band appears at the expected molecular weight of TBC1D4 (~160 kDa) .

• Cross-Reactivity Testing: Test the antibody against recombinant TBC1D1 (a closely related protein) to ensure specificity, as TBC1D1 and TBC1D4 share structural similarities .

These validation steps ensure that experimental results accurately reflect TBC1D4 detection rather than non-specific binding or cross-reactivity with related proteins .

How can TBC1D4 Antibody, Biotin conjugated be used to investigate phosphorylation patterns under different metabolic conditions?

TBC1D4 Antibody, Biotin conjugated can be strategically employed to investigate phosphorylation patterns through the following methodological approach:

• Study Design for Metabolic Conditions: Establish experimental conditions that modulate insulin signaling and AMPK activation, such as exercise protocols (acute vs. chronic), insulin stimulation (physiological meal-induced vs. hyperinsulinemic clamp), or nutrient manipulation (fed vs. fasted state) .

• Combined Immunoprecipitation Strategy: Use the biotin-conjugated TBC1D4 antibody to immunoprecipitate total TBC1D4 protein from sample lysates. This can be followed by Western blotting with site-specific phospho-antibodies targeting known regulatory sites (S318, S341, S588, T642, S666, S704, S751) .

• Multiplex Analysis of Phosphorylation Sites: Different phosphorylation sites on TBC1D4 respond distinctly to insulin and exercise stimuli. Research has identified sites that:

  • Do not respond to either stimulus (S666)

  • Respond to insulin only (S318)

  • Respond to exercise only (S588, S751)

  • Respond to both insulin and exercise (S341, T642, S704)

• Time-Course Analysis: Examine the temporal dynamics of TBC1D4 phosphorylation by collecting samples at multiple time points following stimulation (e.g., immediately post-exercise, 3h post-exercise, etc.) .

• Correlation with Physiological Outcomes: Correlate phosphorylation patterns with glucose uptake measurements to establish functional significance of observed phosphorylation changes .

This approach allows researchers to dissect the complex regulation of TBC1D4 in response to various metabolic stimuli and better understand its role in glucose homeostasis .

What are the methodological considerations when investigating TBC1D4 interactions with 14-3-3 proteins and other binding partners?

Investigating TBC1D4 interactions with 14-3-3 proteins and other binding partners requires careful methodological consideration:

• Co-immunoprecipitation Protocol Optimization:

  • Use the biotin-conjugated TBC1D4 antibody with streptavidin-agarose beads for pull-down experiments

  • Include phosphatase inhibitors (sodium fluoride, sodium pyrophosphate, sodium orthovanadate) in all buffers

  • Optimize salt concentration in washing buffers (typically 100-150 mM NaCl) to maintain specific interactions while reducing background

  • Consider crosslinking approaches for transient interactions

• Phosphorylation-Dependent Interactions: 14-3-3 proteins bind to TBC1D4 in a phosphorylation-dependent manner, primarily at phosphorylated T642. Compare samples under different phosphorylation conditions (basal, insulin-stimulated, AMPK-activated) to capture condition-specific interactions .

• Competitive Binding Experiments: Use synthetic phosphopeptides corresponding to key TBC1D4 phosphorylation sites to compete with native interactions and determine binding specificity .

• Proximity Ligation Assays: For in situ detection of protein-protein interactions, combine the biotin-conjugated TBC1D4 antibody with antibodies against potential binding partners in proximity ligation assays to visualize interactions within intact cells.

• Mutational Analysis: Create phospho-mimetic (S/T to D/E) or phospho-resistant (S/T to A) mutations at key sites to determine the importance of specific phosphorylation events for protein interactions .

This methodological framework allows researchers to dissect the dynamic interactome of TBC1D4 and understand how these interactions mediate its function in glucose uptake regulation .

How does TBC1D4 signaling differ between skeletal muscle and adipose tissue, and what methodological adaptations are required?

TBC1D4 signaling exhibits tissue-specific patterns between skeletal muscle and adipose tissue, requiring methodological adaptations for comprehensive investigation:

• Tissue-Specific Expression Patterns:

  • Skeletal muscle expresses both TBC1D4 and TBC1D1, with TBC1D4 predominating in oxidative fibers and TBC1D1 in glycolytic fibers

  • Adipose tissue expresses primarily TBC1D4 with minimal TBC1D1 expression

  • These expression differences necessitate careful antibody selection and validation in each tissue type

• Methodological Adaptations for Adipose Tissue:

  • Sample preparation: Adipose tissue requires additional defatting steps during homogenization

  • Buffer modifications: Include higher detergent concentrations (1-1.5% Triton X-100) to improve protein extraction

  • Loading controls: Use different loading controls (β-actin for adipose tissue vs. GAPDH for muscle)

  • Immunodetection: May require longer antibody incubation times (overnight at 4°C) for optimal signal

• Differential Phosphorylation Response:

  • Insulin stimulation produces more robust TBC1D4 phosphorylation at T642 in adipocytes compared to muscle

  • Exercise-responsive phosphorylation (S588, S751) is more pronounced in muscle than adipose tissue

  • These differences require targeted phospho-site analysis based on the tissue and stimulus being studied

• Physiological Context Considerations:

  • Skeletal muscle: Consider muscle fiber type composition when interpreting results

  • Adipose tissue: Account for differences between visceral and subcutaneous depots

  • For in vitro studies, primary cells often provide more physiologically relevant responses than immortalized cell lines

These tissue-specific differences in TBC1D4 signaling highlight the importance of tailored methodological approaches when investigating its role in metabolic regulation across different tissues .

What are common challenges in detecting TBC1D4 phosphorylation and how can they be addressed?

Detecting TBC1D4 phosphorylation presents several technical challenges that researchers should be prepared to address:

• Rapid Dephosphorylation During Sample Handling:

  • Challenge: Phosphatases can rapidly dephosphorylate TBC1D4 during sample collection and processing

  • Solution: Immediately snap-freeze samples in liquid nitrogen; use phosphatase inhibitor cocktails containing sodium fluoride (50 mM), sodium pyrophosphate (5 mM), sodium orthovanadate (2 mM), and microcystin (0.1 μM) in all buffers

• Low Signal-to-Noise Ratio:

  • Challenge: Some phosphorylation sites exhibit low stoichiometry, making detection difficult

  • Solution: Enrich TBC1D4 by immunoprecipitation using the biotin-conjugated antibody prior to phospho-specific detection; use enhanced chemiluminescence or fluorescence-based detection systems with increased sensitivity

• Cross-Reactivity with TBC1D1:

  • Challenge: TBC1D1 shares sequence homology with TBC1D4, potentially causing cross-reactivity

  • Solution: Validate antibody specificity using recombinant proteins; consider using TBC1D1-specific knockdown controls; verify molecular weight differences (TBC1D1: ~150 kDa; TBC1D4: ~160 kDa)

• Quantification Challenges:

  • Challenge: Accurate normalization of phosphorylation signals

  • Solution: Always normalize phospho-specific signals to total TBC1D4 protein rather than to loading controls like GAPDH or actin; run multiple technical replicates; use internal calibration curves with phosphorylated recombinant proteins

• Temporal Dynamics:

  • Challenge: Different phosphorylation sites have different kinetics

  • Solution: Design time-course experiments with appropriate time points; consider both rapid (minutes) and delayed (hours) responses for comprehensive analysis

Addressing these challenges through methodological refinements ensures more reliable detection and quantification of TBC1D4 phosphorylation in research applications .

How should researchers interpret changes in TBC1D4 phosphorylation in the context of insulin resistance studies?

When interpreting TBC1D4 phosphorylation data in insulin resistance studies, researchers should consider several key contextual factors:

• Site-Specific Interpretation:

  • Different phosphorylation sites have distinct functional significance

  • Akt-mediated sites (T642, S318, S341) primarily reflect insulin signaling status

  • AMPK-mediated sites (S588, S751) reflect energy status and contraction responses

  • Interpret each site within its specific signaling context rather than assuming uniform responses

• Relative vs. Absolute Changes:

  • Express phosphorylation as a ratio to total TBC1D4 protein

  • Consider both baseline phosphorylation and stimulated response

  • In insulin-resistant states, look for:

    • Altered basal phosphorylation

    • Blunted phosphorylation response to insulin

    • Potentially compensatory increases in exercise-responsive sites

• Correlation with Functional Outcomes:

  • Tie phosphorylation changes to functional measures (glucose uptake, GLUT4 translocation)

  • T642 phosphorylation consistently correlates with insulin-stimulated glucose uptake

  • Impaired T642 phosphorylation may predict defective GLUT4 translocation

• Multi-Tissue Considerations:

  • TBC1D4 regulation may differ between muscle and adipose tissue in the same subject

  • Tissue-specific insulin resistance may show distinct phosphorylation patterns

  • Consider analyzing multiple tissues when possible for comprehensive understanding

• Integrated Pathway Analysis:

  • Interpret TBC1D4 phosphorylation within the broader insulin signaling network

  • Consider upstream regulators (IRS-1, PI3K, Akt, AMPK) and downstream effects

  • Look for disconnects between upstream and downstream signaling events that may indicate pathological adaptations

By applying these interpretative frameworks, researchers can extract meaningful insights from TBC1D4 phosphorylation data in the context of insulin resistance studies .

What are the best practices for analyzing discrepancies between TBC1D4 phosphorylation data and glucose transport measurements?

When researchers encounter discrepancies between TBC1D4 phosphorylation data and glucose transport measurements, the following analytical framework should be applied:

• Technical Verification:

  • Confirm antibody specificity and phospho-site selectivity

  • Verify the timing of measurements (phosphorylation may precede functional changes)

  • Ensure appropriate normalization to total TBC1D4 protein rather than loading controls alone

  • Consider whether samples were collected at optimal time points for both measurements

• Phosphorylation Pattern Analysis:

  • Examine multiple phosphorylation sites, not just single sites

  • TBC1D4 function likely depends on a pattern of phosphorylation across several sites

  • Different combinations of phosphorylated sites may yield similar functional outcomes

  • Some sites may have dominant effects that override others

• Redundancy in Signaling Pathways:

  • TBC1D1 may compensate for altered TBC1D4 function, especially in glycolytic muscle fibers

  • Alternative signaling pathways (e.g., PI3K-dependent but Akt-independent pathways) may bypass TBC1D4

  • Consider analyzing both TBC1D1 and TBC1D4 phosphorylation simultaneously

• Threshold Effects:

  • Phosphorylation may need to reach certain thresholds to trigger functional changes

  • Sub-threshold changes may not translate to measurable glucose transport alterations

  • Consider dose-response relationships rather than single-dose experiments

• Context-Dependent Regulation:

  • Acute vs. chronic conditions may show different relationships between phosphorylation and function

  • Prior exercise history affects the relationship between insulin signaling and glucose uptake

  • Nutritional status modifies the relationship between phosphorylation patterns and functional outcomes

• Resolution of Discrepancies:

  • Design experiments with genetic models (siRNA knockdown, phospho-mimetic mutations)

  • Conduct time-course studies to capture temporal dynamics

  • Combine in vitro and in vivo approaches to bridge mechanistic and physiological relevance

This analytical approach helps researchers resolve apparent discrepancies and develop a more nuanced understanding of TBC1D4's role in regulating glucose transport under various physiological and pathological conditions .

How might TBC1D4 Antibody, Biotin conjugated be utilized in single-cell analysis of skeletal muscle heterogeneity?

Emerging applications of TBC1D4 Antibody, Biotin conjugated in single-cell analysis offer promising approaches to understanding skeletal muscle fiber type heterogeneity:

• Immunofluorescence-Based Single Fiber Analysis:

  • The biotin conjugation enables high-sensitivity detection through streptavidin-fluorophore systems

  • Combine with fiber-type markers (myosin heavy chain isoforms) to correlate TBC1D4 expression/phosphorylation with fiber type

  • Implement tyramide signal amplification (TSA) with the biotin-streptavidin system to detect low-abundance phosphorylation events in single fibers

• Flow Cytometry Applications:

  • Dissociate muscle tissue into single myonuclei or satellite cells

  • Use biotin-conjugated TBC1D4 antibody with streptavidin-fluorophores for intracellular staining

  • Implement phospho-flow cytometry to quantify site-specific phosphorylation at the single-cell level

  • Combine with myosin heavy chain isoform antibodies for fiber-type specific sorting and analysis

• In Situ Proximity Ligation Assay (PLA):

  • Detect TBC1D4 phosphorylation and protein-protein interactions directly in tissue sections

  • The biotin-conjugated antibody can be paired with phospho-specific antibodies in PLA reactions

  • Visualize subcellular localization of phosphorylated TBC1D4 in relation to GLUT4 storage vesicles

  • Quantify differences between fiber types within the same muscle section

• Single-Cell Western Blotting:

  • Apply microfluidic approaches to analyze individual muscle fibers or isolated cells

  • Use biotin-conjugated antibody with chemiluminescent or fluorescent streptavidin detection

  • Quantify TBC1D4 expression and phosphorylation variations between individual cells

  • Correlate with metabolic phenotypes at the single-cell level

These emerging single-cell applications offer unprecedented resolution for understanding the heterogeneous regulation of glucose metabolism across different muscle fiber types and may reveal novel insights into insulin resistance mechanisms .

What methodological considerations should be addressed when investigating the interplay between TBC1D1 and TBC1D4 in glucose metabolism?

Investigating the interplay between TBC1D1 and TBC1D4 requires specialized methodological approaches to disentangle their distinct yet overlapping roles:

• Tissue and Fiber Type Considerations:

  • TBC1D1 predominates in glycolytic fibers while TBC1D4 is more abundant in oxidative fibers

  • Select appropriate muscle groups for study (e.g., soleus for TBC1D4, EDL for TBC1D1 in rodents)

  • In human studies, ensure muscle biopsies contain sufficient representation of both fiber types

  • Consider fiber type composition when interpreting results from mixed muscle samples

• Protein-Specific Immunoprecipitation:

  • Use highly specific antibodies to selectively immunoprecipitate each protein

  • The biotin-conjugated TBC1D4 antibody can be used with streptavidin beads for clean pull-downs

  • Verify absence of cross-precipitation by immunoblotting precipitates for both proteins

  • Consider sequential immunoprecipitation to deplete one protein before analyzing the other

• Phosphorylation Profiling:

  • Develop parallel phosphorylation profiles for both proteins under identical conditions

  • Compare phosphorylation patterns in response to insulin, AICAR (AMPK activator), and exercise

  • Distinguish shared vs. protein-specific phosphorylation responses

  • Correlate with downstream functional readouts (GLUT4 translocation, glucose uptake)

• Gene Silencing Approaches:

  • Design siRNA/shRNA strategies for selective knockdown of each protein

  • Implement single and double knockdown experiments to assess compensation

  • Evaluate phosphorylation of the remaining protein after knockdown of its counterpart

  • Quantify functional consequences of single vs. double knockdown

• Substrate Specificity Analysis:

  • Investigate Rab GTPase specificity differences between TBC1D1 and TBC1D4

  • Determine whether they regulate distinct or overlapping pools of GLUT4 vesicles

  • Assess differential effects on glucose vs. fatty acid metabolism

  • Consider potential differences in compartmentalization and subcellular localization

These methodological considerations enable researchers to dissect the complementary and sometimes redundant roles of TBC1D1 and TBC1D4 in regulating glucose metabolism, particularly in the context of fiber type-specific metabolic regulation .

How can TBC1D4 phosphorylation analysis contribute to personalized exercise prescription in insulin resistance?

TBC1D4 phosphorylation analysis offers promising applications for developing personalized exercise prescriptions in insulin resistance, with several methodological considerations:

• Individual Phosphorylation Fingerprinting:

  • Establish baseline phosphorylation profiles at key sites (T642, S588, S751) in muscle biopsies

  • Determine acute responses to standardized exercise bouts of varying intensity and duration

  • Identify individual-specific phosphorylation patterns that predict improved glucose handling

  • Use the biotin-conjugated antibody with site-specific phospho-antibodies for comprehensive profiling

• Exercise Protocol Optimization Framework:

  • Design a testing battery of different exercise protocols (high-intensity interval, moderate continuous, resistance)

  • Measure site-specific phosphorylation responses to each protocol

  • Correlate acute phosphorylation changes with improvements in glucose tolerance

  • Develop predictive models that match optimal exercise prescription to individual phosphorylation responses

• Genetic Variant Stratification:

  • Screen for known TBC1D4 gene variants associated with altered protein function (e.g., Arg684Ter variant)

  • Stratify phosphorylation responses by genotype

  • Determine whether specific exercise modalities overcome genotype-associated signaling defects

  • Develop genotype-guided exercise prescription frameworks

• Monitoring Methodology Development:

  • Establish minimally invasive protocols (microbiopsy techniques)

  • Develop standardized analysis pipelines for rapid sample processing

  • Create reference databases of phosphorylation responses in different populations

  • Implement serial monitoring to track adaptive responses to training programs

• Integration with Clinical Outcomes:

  • Correlate TBC1D4 phosphorylation patterns with clinically relevant outcomes

  • Develop predictive models that use acute phosphorylation responses to forecast long-term benefits

  • Create decision-support algorithms for exercise prescription

  • Validate with randomized controlled trials comparing standard vs. phosphorylation-guided exercise prescription

This approach leverages molecular signaling data to move beyond one-size-fits-all exercise recommendations, potentially improving adherence and effectiveness of lifestyle interventions for insulin resistance by matching exercise protocols to individual molecular response patterns .

What current evidence exists regarding the role of TBC1D4 in conditions beyond type 2 diabetes?

Recent research has expanded our understanding of TBC1D4's role beyond type 2 diabetes to several other physiological and pathological conditions:

• Cardiovascular Health:

  • TBC1D4 phosphorylation status correlates with endothelial function

  • Impaired TBC1D4 signaling may contribute to cardiovascular complications independent of glycemic control

  • Exercise-induced improvements in vascular function may partially operate through TBC1D4-mediated pathways

  • Methodological approach: Correlate TBC1D4 phosphorylation in muscle biopsies with flow-mediated dilation measurements

• Neurodegenerative Conditions:

  • Emerging evidence suggests TBC1D4 plays a role in neuronal glucose metabolism

  • Brain insulin resistance may involve altered TBC1D4 phosphorylation patterns

  • TBC1D4 may influence amyloid processing and tau phosphorylation

  • Methodological approach: Use the biotin-conjugated antibody for immunohistochemical analysis of brain tissue sections

• Cancer Metabolism:

  • Altered TBC1D4 expression has been observed in several cancer types

  • Cancer cells may exploit TBC1D4-mediated GLUT4 trafficking to enhance glucose uptake

  • TBC1D4 phosphorylation status may influence tumor response to metabolic stress

  • Methodological approach: Compare TBC1D4 phosphorylation in tumor vs. adjacent normal tissue; correlate with FDG-PET imaging data

• Aging-Related Insulin Resistance:

  • Age-related declines in muscle insulin sensitivity correlate with reduced TBC1D4 phosphorylation capacity

  • Exercise training partially reverses age-related defects in TBC1D4 signaling

  • Methodological approach: Age-stratified analysis of TBC1D4 phosphorylation responses to insulin and exercise stimulation

• Inflammatory Conditions:

  • Preliminary studies found no significant changes in TBC1D4 expression when adipocytes were treated with inflammatory cytokines and adipokines

  • This suggests that inflammation may impair insulin signaling through mechanisms independent of TBC1D4 expression levels

  • Methodological approach: Measure both expression and phosphorylation status in response to inflammatory stimuli

These emerging research areas highlight the diverse physiological roles of TBC1D4 beyond glucose metabolism and suggest new applications for TBC1D4 Antibody, Biotin conjugated in investigating these conditions .

What novel methodological approaches could enhance the study of TBC1D4 phosphorylation dynamics?

Several innovative methodological approaches are emerging that could significantly enhance our understanding of TBC1D4 phosphorylation dynamics:

• CRISPR-Mediated Endogenous Tagging:

  • Generate cell lines or animal models with endogenous TBC1D4 tagged with fluorescent proteins

  • Combine with the biotin-conjugated antibody for live-cell imaging and fixed-cell analysis

  • Monitor real-time trafficking of TBC1D4 in response to insulin or exercise stimuli

  • Correlate subcellular localization with phosphorylation status through fixed-time-point analysis

• Mass Spectrometry-Based Phosphoproteomics:

  • Implement targeted phosphoproteomics to quantify multiple phosphorylation sites simultaneously

  • Use the biotin-conjugated antibody for affinity purification before mass spectrometry analysis

  • Determine phosphorylation stoichiometry at each site under various conditions

  • Identify novel phosphorylation sites beyond those currently recognized

  • Create comprehensive phosphorylation signatures in health and disease states

• Biosensor Development:

  • Design FRET-based biosensors to monitor TBC1D4 phosphorylation in real-time

  • Develop biosensors specific for key phosphorylation sites (T642, S588)

  • Monitor phosphorylation kinetics in living cells following various stimuli

  • Correlate with GLUT4 translocation dynamics using dual-reporter systems

• Optical Tissue Clearing Techniques:

  • Apply advanced tissue clearing methods (CLARITY, iDISCO) to whole muscle samples

  • Use the biotin-conjugated antibody with fluorescent streptavidin for 3D imaging

  • Map fiber type-specific TBC1D4 distribution and phosphorylation in intact muscles

  • Analyze spatial heterogeneity of TBC1D4 signaling within and between muscle fibers

• Single-Molecule Tracking:

  • Implement super-resolution microscopy with the biotin-conjugated antibody

  • Track individual TBC1D4 molecules in relation to GLUT4 vesicles

  • Determine how phosphorylation affects molecular dynamics and protein-protein interactions

  • Correlate molecular behavior with functional outcomes in glucose transport

These innovative approaches promise to provide unprecedented spatial and temporal resolution for studying TBC1D4 phosphorylation dynamics, potentially revealing new regulatory mechanisms and therapeutic targets for metabolic disorders .

How might multi-omics integration enhance our understanding of TBC1D4 regulation in metabolic health?

Multi-omics integration offers powerful approaches to contextualize TBC1D4 regulation within broader metabolic networks:

• Integrated Phosphoproteomics and Metabolomics:

  • Correlate TBC1D4 phosphorylation patterns with global metabolite profiles

  • Identify metabolic signatures associated with specific TBC1D4 phosphorylation states

  • Use the biotin-conjugated antibody for TBC1D4 enrichment prior to phosphoproteomic analysis

  • Develop predictive models linking TBC1D4 phosphorylation to metabolic outcomes

  • Methodological approach: Parallel analysis of muscle biopsies for phosphoproteomics and targeted metabolomics

• Transcriptomics-Proteomics Integration:

  • Compare TBC1D4 mRNA expression with protein abundance across tissues and conditions

  • Identify factors that regulate TBC1D4 at transcriptional and post-transcriptional levels

  • Discover gene networks that co-regulate with TBC1D4 under various physiological states

  • Methodological approach: RNA-seq combined with quantitative proteomics using the biotin-conjugated antibody for protein validation

• Epigenetic Regulation Analysis:

  • Investigate DNA methylation and histone modifications at the TBC1D4 gene locus

  • Determine how exercise training or insulin resistance affects epigenetic regulation

  • Correlate epigenetic modifications with TBC1D4 expression and phosphorylation

  • Methodological approach: Chromatin immunoprecipitation sequencing (ChIP-seq) combined with bisulfite sequencing and protein analysis

• Single-Cell Multi-Omics:

  • Apply single-cell RNA-seq with targeted proteomics in skeletal muscle

  • Characterize cell-specific TBC1D4 regulation within heterogeneous tissues

  • Identify cell populations with distinct TBC1D4 signaling profiles

  • Methodological approach: Single-cell sequencing with protein detection using index sorting and the biotin-conjugated antibody

• Network Analysis and Systems Biology:

  • Construct protein-protein interaction networks centered on TBC1D4

  • Model how phosphorylation alters interaction dynamics within the network

  • Simulate perturbations to predict therapeutic targets

  • Validate key network nodes using the biotin-conjugated antibody for co-immunoprecipitation

  • Methodological approach: Interactome analysis through affinity purification-mass spectrometry with computational modeling