TBC1D4 Antibody, FITC conjugated

What is TBC1D4 and what is its biological significance?

TBC1D4, also known as AS160 (Akt Substrate of 160 kDa), functions as a Rab GTPase-activating protein (RabGAP) that plays a critical role in glucose transporter 4 (GLUT4) trafficking. This protein serves as a convergence point for insulin- and exercise-induced signaling in skeletal muscle . TBC1D4 contains multiple phosphorylation sites, with Thr642 being particularly important for insulin-stimulated glucose uptake. Research has shown that TBC1D4 loss-of-function mutations in humans are associated with an increased risk of type 2 diabetes .

The protein functions by maintaining Rab proteins in an inactive state under basal conditions, effectively preventing GLUT4 translocation to the cell surface. Upon insulin stimulation, TBC1D4 becomes phosphorylated, which inhibits its RabGAP activity, allowing Rab proteins to become activated and facilitate GLUT4 translocation to the plasma membrane .

How does a FITC-conjugated TBC1D4 antibody differ from unconjugated versions?

FITC (Fluorescein Isothiocyanate) conjugation provides direct fluorescent labeling of the TBC1D4 antibody, eliminating the need for secondary antibody detection steps. The FITC fluorophore is covalently linked to the primary antibody using established crosslinking protocols . While unconjugated TBC1D4 antibodies require a secondary detection system (such as a fluorescently-labeled or enzyme-linked secondary antibody), FITC-conjugated antibodies enable direct visualization through fluorescence microscopy with appropriate FITC filters (excitation ~495 nm, emission ~520 nm) .

Key considerations when choosing between conjugated and unconjugated formats include:

What are the optimal conditions for using TBC1D4 Antibody, FITC conjugated in immunofluorescence experiments?

When using FITC-conjugated TBC1D4 antibodies for immunofluorescence, researchers should follow these methodological guidelines for optimal results:

• Cell preparation and fixation:

  • Fix cells with 4% paraformaldehyde for 10 minutes at room temperature

  • Permeabilize with 0.1% Triton X-100 for 5 minutes

  • Wash thoroughly with PBS (3 × 5 minutes)

• Blocking and antibody incubation:

  • Block with PBS containing 10% fetal bovine serum (FBS) for 20 minutes at room temperature

  • Dilute FITC-conjugated TBC1D4 antibody 1:500 in PBS/10% FBS

  • Incubate for 1 hour at room temperature in the dark

  • Wash cells 2 × 5 minutes with PBS

• Imaging considerations:

  • Use a fluorescence microscope equipped with appropriate FITC filters

  • Minimize exposure to light during all steps to prevent photobleaching

  • For co-localization studies, ensure filter sets have minimal spectral overlap

For skeletal muscle tissue sections, additional optimization may be required, including longer antibody incubation times (overnight at 4°C) and more extensive blocking steps to reduce non-specific binding.

How can I design experiments to investigate TBC1D4 phosphorylation states using FITC-conjugated phospho-specific antibodies?

Designing experiments to study TBC1D4 phosphorylation requires careful consideration of stimulation conditions, controls, and analysis methods:

• Stimulation conditions:

  • Insulin stimulation: 100 nM insulin for 10-30 minutes

  • Exercise or contraction mimetics: AICAR (AMPK activator) or increased intracellular Ca²⁺

  • Combined stimuli: Pretreatment with AICAR followed by insulin can reveal important regulatory transitions

• Essential controls:

  • Unstimulated baseline samples

  • Phosphatase treatment controls (to confirm phospho-specificity)

  • Knockdown or knockout controls (TBC1D4-KO samples to verify antibody specificity)

  • Competing peptide controls

• Analytical approaches:

  • Co-localization with total TBC1D4 antibody (different fluorophore)

  • Time-course analysis to capture dynamic phosphorylation events

  • Quantitative analysis using standardized imaging parameters

For phospho-specific detection, use antibodies targeting key phosphorylation sites such as pThr642-TBC1D4, which is the primary insulin-responsive phosphorylation site . The phosphorylation status of TBC1D4 at Thr642 correlates strongly with insulin-stimulated GLUT4 translocation and can be used as a biomarker for insulin sensitivity.

How can FITC-conjugated TBC1D4 antibodies be used to investigate protein-protein interactions in the GLUT4 trafficking pathway?

FITC-conjugated TBC1D4 antibodies can be leveraged for sophisticated studies of protein-protein interactions using these methodological approaches:

• Co-immunoprecipitation coupled with fluorescence detection:

  • Use TBC1D4 antibodies to immunoprecipitate the protein complex

  • Analyze interaction partners through proteomic approaches

  • Recent studies identified 76 proteins as candidate TBC1D4 interactors, including 14-3-3 proteins and α-actinin-4 (ACTN4)

• Proximity ligation assay (PLA):

  • Combine FITC-conjugated TBC1D4 antibody with antibodies against potential interaction partners

  • Visualize interactions as fluorescent spots when proteins are within 40 nm

  • Quantify interaction frequency under different physiological conditions

• FRET (Förster Resonance Energy Transfer) analysis:

  • Use FITC as a donor fluorophore and pair with acceptor fluorophore-labeled antibodies against interaction partners

  • Measure energy transfer as evidence of molecular proximity

  • Particularly useful for studying dynamic interactions with 14-3-3 proteins after insulin stimulation

A significant finding from recent research is that several TBC1D4 interactions are regulated by insulin, with 12 of the 76 identified interacting proteins showing insulin-dependent binding patterns . This suggests that TBC1D4 serves as a dynamic scaffold for assembling signaling complexes involved in glucose homeostasis.

What are the experimental approaches to study the cooperative governance between TBC1D1 and TBC1D4 using fluorescently labeled antibodies?

Studying the cooperative relationship between TBC1D1 and TBC1D4 requires specialized experimental approaches:

• Dual immunofluorescence labeling:

  • Use FITC-conjugated TBC1D4 antibody together with a differently labeled TBC1D1 antibody

  • Quantify co-localization in different subcellular compartments

  • Analyze changes in co-localization patterns following various stimuli

• Knockdown/knockout models with reconstitution:

  • Generate cells with knockdown of either TBC1D1, TBC1D4, or both proteins

  • Reconstitute with wild-type or mutant versions to assess functional cooperation

  • Research has shown that when both RabGAPs are present, TBC1D1 functionally dominates AS160 (TBC1D4), and stimuli-inducible GLUT4 release relies on TBC1D1-evoking proximal stimuli

• Site-specific phosphorylation analysis:

  • Key phosphorylation sites include Thr642 on TBC1D4 and Ser237/Thr596 on TBC1D1

  • Synergizing actions rely on the phosphotyrosine-binding 1 (PTB1) and calmodulin-binding domains of TBC1D1

  • Use specific antibodies against these phosphosites to track activation status

• GLUT4 trafficking analysis with varying expression ratios:

  • Studies using varying expression ratios of TBC1D1 to TBC1D4 revealed that cells with increased intensity ratio of TBC1D1 relative to TBC1D4 showed dampening of insulin-responsive increases in GLUT4 movement

The research reveals an emerging cooperative governance relying on multiple regulatory nodes of both TBC1D1 and TBC1D4 that together play a key role in properly deciphering biochemical signals into physical GLUT4 release processes in response to insulin, exercise, and the two in combination .

How can I address non-specific binding when using FITC-conjugated TBC1D4 antibodies?

Non-specific binding is a common challenge with immunofluorescence studies using FITC-conjugated antibodies. To address this issue:

• Optimize blocking conditions:

  • Extend blocking time to 30-60 minutes using PBS with 10% FBS

  • Consider alternative blocking agents such as 5% BSA or 5% normal serum from the same species as the secondary antibody

  • For tissues with high autofluorescence, include 0.1-0.3% Triton X-100 in blocking buffer

• Validate antibody specificity:

  • Use TBC1D4-knockout controls to establish baseline non-specific signal

  • In published studies, researchers compared TBC1D4 immunoprecipitation from TBC1D4-WT and whole-body TBC1D4-KO mice to identify true interactors versus non-specific binding

  • Employ competing peptide controls to confirm binding specificity

• Antibody dilution optimization:

  • Test serial dilutions (1:250, 1:500, 1:1000) to identify optimal signal-to-noise ratio

  • Consider using affinity-purified antibodies (>95% purity) for reduced background

• Technical considerations:

  • Include proper negative controls (omission of primary antibody)

  • Use freshly prepared paraformaldehyde for fixation

  • Consider photobleaching of background autofluorescence before imaging

Research has shown that of 149 initially identified human TBC1D4 interactors, 73 were later attributed to antibody cross-reactivity and excluded from analysis , highlighting the importance of rigorous specificity validation.

What are the best practices for maintaining FITC-conjugated antibody stability and performance over time?

Maintaining optimal performance of FITC-conjugated TBC1D4 antibodies requires careful handling:

• Storage conditions:

  • Store at -20°C or -80°C in the dark

  • Avoid repeated freeze-thaw cycles

  • For working solutions, store at 4°C for no more than one week

• Buffer composition effects:

  • Most commercial preparations contain preservatives such as 0.03% Proclin 300

  • Typical formulations include 50% Glycerol and 0.01M PBS at pH 7.4

  • Do not add sodium azide to peroxidase-conjugated antibodies

• Light exposure management:

  • FITC is particularly susceptible to photobleaching

  • Wrap tubes in aluminum foil

  • Minimize exposure to light during all experimental steps

  • Work in reduced ambient lighting when possible

• Quality control checks:

  • Periodically test antibody performance on positive control samples

  • Include standardized controls in each experiment to track antibody performance over time

  • Consider preparing single-use aliquots to minimize freeze-thaw cycles

The data below shows typical antibody performance decline under different storage conditions over time:

How can I quantitatively analyze TBC1D4 phosphorylation dynamics using fluorescence microscopy data?

For rigorous quantitative analysis of TBC1D4 phosphorylation using fluorescence microscopy:

• Image acquisition standardization:

  • Use identical exposure settings, gain, and offset across all experimental conditions

  • Acquire multiple fields (>10) per condition to account for cellular heterogeneity

  • Include reference standards for fluorescence intensity calibration

• Image analysis approaches:

  • Measure mean fluorescence intensity within defined cellular regions

  • For phospho-specific analysis, normalize to total TBC1D4 signal

  • Consider subcellular distribution analysis (membrane vs. cytosolic fractions)

  • Research has shown that TBC1D4 translocates from membranes to cytosol upon insulin stimulation

• Statistical analysis strategies:

  • For comparing conditions (e.g., basal vs. insulin-stimulated):

    • Paired t-tests for matched sample comparisons

    • Two-way repeated-measures ANOVA for multiple condition analysis

  • For time-course experiments, consider area under the curve analysis

• Visualization methods:

  • Present data as fold-change relative to basal conditions

  • Show representative images alongside quantitative data

  • Use consistent pseudocoloring across all figures

In published research, analysis of insulin-induced percentage increases in the mean speed of GLUT4 movement (resulting from GLUT4 liberation) plotted versus the relative ratio of TBC1D1 to TBC1D4 revealed a negative correlation between these values , demonstrating how quantitative analysis can reveal important regulatory relationships.

How do I interpret contradictory results between TBC1D4 phosphorylation states and GLUT4 translocation outcomes?

Contradictory results between TBC1D4 phosphorylation and GLUT4 translocation often reflect the complex regulatory network. When facing such contradictions:

• Consider temporal dynamics:

  • TBC1D4 phosphorylation typically precedes detectable GLUT4 translocation

  • Research shows TBC1D4 phosphorylation can be detected within 5 minutes of insulin stimulation, while GLUT4 translocation may take 10-30 minutes to reach maximum

  • Sequential sampling may reconcile apparently contradictory results

• Account for cooperative regulation with TBC1D1:

  • TBC1D1 can functionally dominate TBC1D4 when both are present

  • The relative expression ratio of TBC1D1:TBC1D4 influences insulin responsiveness

  • Mutation studies have shown that phosphorylation of both proteins at specific sites (Thr642 on TBC1D4 and Ser237/Thr596 on TBC1D1) is necessary for proper GLUT4 release

• Evaluate multiple phosphorylation sites:

  • While Thr642 is the dominant insulin-responsive site on TBC1D4, additional sites (Ser318, Ser588, Ser704) also contribute

  • Incomplete phosphorylation across all sites may lead to partial activation

  • Single-site analysis may miss important regulatory events

• Consider compartmentalization effects:

  • Research has demonstrated that TBC1D4 association with membranes is required for its inhibitory action under basal conditions, but its insulin-dependent dissociation is not required for GLUT4 translocation

  • This suggests that phosphorylation is the primary regulatory mechanism, while translocation may be a secondary effect

When interpreting contradictory results, it's important to note that mutations in the PTB1 domain of TBC1D1, including an obesity-related R125W mutation, prevent the regulatory mode shift for insulin responsiveness acquisition, even in the presence of TBC1D4 .

How can FITC-conjugated TBC1D4 antibodies be used to investigate the role of TBC1D4 in pathological conditions?

FITC-conjugated TBC1D4 antibodies offer valuable tools for investigating pathological conditions:

• Type 2 diabetes research applications:

  • Compare TBC1D4 phosphorylation patterns between healthy subjects and patients with diabetes

  • Research has shown that TBC1D4 loss-of-function mutations in human skeletal muscle are associated with increased risk of type 2 diabetes

  • Analyze exercise-induced changes in TBC1D4 phosphorylation to understand exercise resistance mechanisms

• Obesity studies:

  • Investigate the relationship between TBC1D1 variants (e.g., R125W obesity-related mutation) and TBC1D4 function

  • Examine adipose tissue-specific changes in TBC1D4 localization and phosphorylation

  • Correlate findings with metabolic parameters and insulin sensitivity indices

• Experimental disease models:

  • Use diet-induced obesity models to track changes in TBC1D4 regulation over time

  • Apply FITC-conjugated antibodies in tissue section analysis for regional differences

  • Combine with metabolic phenotyping to establish correlative relationships

• Therapeutic intervention assessment:

  • Monitor TBC1D4 phosphorylation as a biomarker for insulin-sensitizing drug efficacy

  • Track changes during exercise interventions or weight loss programs

  • Use as a surrogate endpoint in preclinical intervention studies

Recent research indicates that TBC1D4 and TBC1D1 cooperatively regulate stimuli-responsive GLUT4-releasing activities , suggesting that comprehensive analysis of both proteins may provide deeper insights into pathological mechanisms underlying insulin resistance.

What are the latest methodological advances in multiplex imaging combining TBC1D4 detection with other signaling pathway components?

Recent advances in multiplex imaging have expanded the capabilities for studying TBC1D4 in complex signaling networks:

• Spectrally resolved multiplexing:

  • Combine FITC-conjugated TBC1D4 antibodies with other fluorophores (e.g., Cy3, Cy5, Alexa dyes)

  • Utilize linear unmixing algorithms to separate overlapping signals

  • Enable simultaneous detection of TBC1D4 with upstream regulators (Akt, AMPK) and downstream effectors (Rab proteins, GLUT4)

• Cyclic immunofluorescence approaches:

  • Perform sequential staining-imaging-bleaching cycles

  • Allow for detection of >10 targets on the same sample

  • Particularly valuable for mapping comprehensive TBC1D4 interaction networks identified in proteomic studies

• Super-resolution microscopy applications:

  • STORM/PALM techniques achieve 20-30 nm resolution to visualize TBC1D4 nano-domains

  • Single-molecule tracking using quantum dot-conjugated antibodies

  • Research has employed quantum dot-based single molecule analysis to quantitatively describe the "states" of GLUT4 behavior (static or released) with TBC1D4 regulation

• Proximity-based methods:

  • BiFC (Bimolecular Fluorescence Complementation) for direct protein interaction verification

  • FRET-FLIM (Fluorescence Lifetime Imaging) for quantitative interaction measurements

  • Particularly useful for studying dynamic associations between TBC1D4 and 14-3-3 proteins

The integration of these advanced imaging methods with targeted perturbations (such as the use of TBC1D4-KO models or expression of mutant forms) offers unprecedented insights into the spatiotemporal dynamics of glucose homeostasis regulation at the molecular level.