TBC1D1 Antibody, HRP conjugated

What is TBC1D1 and why is it important in metabolic research?

TBC1D1 is a Rab-GTPase-Activating Protein (Rab-GAP) closely related to AS160 (TBC1D4) that plays a critical role in regulating glucose uptake in muscle cells. It functions by maintaining Rab proteins in the inactive GDP-bound form, which prevents GLUT4 translocation in the absence of stimuli. TBC1D1 contains multiple phosphorylation sites for the Ser/Thr kinases Akt and AMPK, making it a key convergence point for insulin and energy-sensing pathways in the regulation of cellular glucose uptake . Its importance in metabolic research stems from its role in glucose homeostasis and potential implications in metabolic disorders like diabetes and obesity.

How does TBC1D1 expression vary across different tissue types?

TBC1D1 expression exhibits significant tissue specificity, with severalfold higher expression in skeletal muscles compared to all other tissues. Within muscle groups, there are notable differences: TBC1D1 mRNA is many-fold greater in fast-twitch muscles (such as tibialis anterior) than in slow-twitch muscles (like soleus), where AS160 is more prevalent . This tissue-specific expression pattern suggests specialized functions of TBC1D1 in different muscle fiber types and metabolic conditions, which researchers should consider when designing tissue-specific studies.

What are the key protein interactions of TBC1D1 that can be studied using its antibodies?

TBC1D1 engages in stable associations with several proteins involved in cellular trafficking. Its PTB domains interact with Rab regulatory proteins including MICAL1 and EHBP1L1, the calcium pump SERCA1, and regulatory proteins VPS13A and VPS13C . Notably, TBC1D1 exhibits isoform-specific interaction with AMPK, associating with heterotrimers containing α1 but not α2 subunits . These interactions can be studied through co-immunoprecipitation experiments using TBC1D1 antibodies, followed by western blotting or mass spectrometry to identify binding partners and characterize the nature of these associations.

What are the optimal conditions for using TBC1D1 Antibody, HRP conjugated in Western blotting?

For optimal Western blotting with HRP-conjugated TBC1D1 antibodies, researchers should consider the following protocol: (1) Prepare protein samples with appropriate lysis buffers containing phosphatase inhibitors to preserve phosphorylation status; (2) Use reducing conditions for SDS-PAGE; (3) Transfer proteins to PVDF or nitrocellulose membranes at 100V for 60-90 minutes in cold transfer buffer; (4) Block with 5% non-fat dry milk or BSA in TBST for 1 hour; (5) Apply the HRP-conjugated TBC1D1 antibody at a 1:1000 to 1:5000 dilution (optimize for your specific antibody); (6) Incubate overnight at 4°C or for 2 hours at room temperature; (7) Wash thoroughly with TBST; (8) Develop using enhanced chemiluminescence substrate without the need for secondary antibody incubation since the primary antibody is already HRP-conjugated .

How should researchers design immunoprecipitation experiments to study TBC1D1 phosphorylation?

When designing immunoprecipitation experiments to study TBC1D1 phosphorylation, researchers should:

• Prepare tissue/cell lysates (typically ~5 mg/ml) in a buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 1 mM EDTA

  • 1 mM EGTA

  • 1% Triton X-100

  • Protease and phosphatase inhibitor cocktails

• Pre-clear lysates with Protein G beads to reduce non-specific binding

• Immunoprecipitate TBC1D1 using specific antibodies coupled to Protein G beads

• For phosphorylation studies, use phospho-specific antibodies or the PAS antibody which detects phosphorylated Akt substrate motifs

• Resuspend immunoprecipitated complexes in appropriate enzyme buffer with DTT and BSA

• For in vitro kinase assays, combine immunoprecipitated TBC1D1 with kinase activation buffer and diluted enzymes at a ratio of 1:2:1:1

• Analyze phosphorylation by immunoblotting with phospho-specific or PAS antibodies

This approach allows for targeted enrichment of TBC1D1 from complex biological samples and specific analysis of its phosphorylation state.

What controls should be included when validating TBC1D1 antibody specificity?

When validating TBC1D1 antibody specificity, researchers should include these essential controls:

• Positive control: Lysates from tissues known to express high levels of TBC1D1 (such as skeletal muscle, particularly tibialis anterior)

• Negative control: Lysates from either:

  • Tissues with minimal TBC1D1 expression

  • TBC1D1 knockout or knockdown samples

  • Pre-absorption of antibody with immunizing peptide

• Peptide competition assay: Compare antibody binding with and without pre-incubation with the immunizing peptide (566-718AA of human TBC1D1)

• Cross-reactivity assessment: Test against samples containing related proteins (especially AS160/TBC1D4) to ensure specificity

• Molecular weight verification: Confirm detection at the expected molecular weight (~160 kDa)

• Isotype control: Use a non-specific IgG from the same host species (rabbit) to demonstrate specific binding

These controls help establish confidence in antibody specificity, particularly important given the structural similarities between TBC1D1 and AS160/TBC1D4.

How can researchers distinguish between TBC1D1 and AS160 (TBC1D4) in experimental data?

Distinguishing between TBC1D1 and AS160 (TBC1D4) requires a strategic approach due to their structural similarities:

• Antibody selection: Use antibodies raised against non-conserved regions. The TBC1D1 antibody targeting amino acids 566-718 provides good specificity .

• Molecular weight discrimination: While both migrate at approximately 160 kDa, careful SDS-PAGE optimization can reveal slight mobility differences.

• Sequential immunodepletion: For complex samples, deplete AS160 first using specific antibodies (targeting C-terminal or splice-specific regions) before TBC1D1 immunoprecipitation .

• Tissue-specific expression patterns: Leverage the differential expression - TBC1D1
  is many-fold higher in fast-twitch muscles (tibialis anterior) while AS160 is more abundant in slow-twitch muscles (soleus) .

• Mass spectrometry: For definitive identification, excise bands after SDS-PAGE and analyze by LC-MS/MS to identify unique peptides specific to each protein .

• Phosphorylation site analysis: Target phosphorylation sites unique to each protein (e.g., Ser237 for TBC1D1 vs. Thr642 for AS160) .

This multi-faceted approach enables reliable discrimination between these closely related Rab-GAPs in experimental settings.

What are common pitfalls when analyzing TBC1D1 phosphorylation data using HRP-conjugated antibodies?

When analyzing TBC1D1 phosphorylation data using HRP-conjugated antibodies, researchers should be aware of these common pitfalls:

• Signal saturation: HRP-conjugated antibodies can produce very strong signals that saturate detection systems, leading to inaccurate quantification. Use multiple exposure times and ensure signals are within the linear range of detection.

• Phosphatase activity during sample preparation: Inadequate phosphatase inhibition can lead to dephosphorylation and underestimation of phosphorylation levels. Use fresh, comprehensive phosphatase inhibitor cocktails in all buffers.

• Phospho-specificity verification: Not all phospho-specific antibodies are equally specific; validate using appropriate controls such as phosphatase-treated samples or AMPK-stimulated versus non-stimulated conditions .

• Context-dependent phosphorylation: TBC1D1 contains multiple phosphorylation sites with different kinetics and stimulus dependencies. The isoform-specific association with AMPKα1 affects phosphorylation patterns, particularly at Ser237 .

• Cross-reactivity with AS160 phospho-sites: Some phospho-specific antibodies may cross-react with similar phosphorylation sites on AS160, complicating interpretation.

• Storage degradation: HRP activity can diminish over time or with repeated freeze-thaw cycles, leading to inconsistent results across experiments .

Addressing these issues through careful experimental design and appropriate controls ensures more reliable phosphorylation data interpretation.

How should researchers normalize and quantify TBC1D1 levels in Western blot experiments?

For accurate normalization and quantification of TBC1D1 levels in Western blot experiments:

• Loading control selection:

  • Use housekeeping proteins like GAPDH, β-actin, or α-tubulin for whole cell lysates

  • For subcellular fractions, use compartment-specific markers (e.g., Na+/K+ ATPase for plasma membrane)

  • Consider total protein normalization methods like Ponceau S or REVERT total protein stain for more accurate normalization

• Technical considerations:

  • Load samples within the linear range of detection (typically 10-50 μg total protein)

  • Run a standard curve with known amounts of recombinant TBC1D1 for absolute quantification

  • Process all samples for comparison simultaneously on the same gel/blot

• Quantification methodology:

  • Use calibrated densitometry software (ImageJ, Image Lab, etc.)

  • Subtract local background for each lane

  • For phosphorylation studies, calculate the ratio of phosphorylated to total TBC1D1

  • For expression studies, normalize to appropriate loading controls

• Statistical analysis:

  • Perform experiments in biological triplicates at minimum

  • Apply appropriate statistical tests based on experimental design

  • Report relative expression as fold-change compared to control conditions

This systematic approach ensures reliable and reproducible quantification of TBC1D1 protein levels across experimental conditions.

How can researchers effectively use TBC1D1 Antibody, HRP conjugated in SILAC-based proteomics experiments?

For effective integration of HRP-conjugated TBC1D1 antibodies in SILAC-based proteomics:

• Experimental design for SILAC:

  • Culture cells in "light" (normal amino acids), "medium" (13C6-Arg/Lys), and "heavy" (13C615N4-Arg/13C615N2-Lys) media for at least 5 cell doublings

  • Apply different experimental conditions to each SILAC state (e.g., control, insulin stimulation, AMPK activation)

  • Verify >95% incorporation of labeled amino acids by mass spectrometry

• Sample preparation workflow:

  • Combine equal amounts of protein from each SILAC condition

  • Immunoprecipitate TBC1D1 using specific antibodies

  • For interactome studies, use constructs expressing TBC1D1 domains (e.g., PTB domains)

  • Separate by SDS-PAGE and process for mass spectrometry analysis

• Verification steps:

  • Confirm immunoprecipitation efficiency with a small aliquot using Western blot

  • Use HRP-conjugated TBC1D1 antibody at 1:1000 dilution for verification

  • Include GFP-trap based immunoprecipitations for tagged constructs

• Data analysis considerations:

  • Analyze SILAC ratios to determine differential interactions or modifications

  • Apply appropriate statistical methods to determine significance

  • Validate key interactions by orthogonal methods (co-IP, Western blot)

This approach has successfully identified novel TBC1D1-interacting proteins, including AMPK heterotrimers containing α1 subunits and Rab regulatory proteins .

What is the significance of the R125W mutation in TBC1D1 and how can it be studied?

The R125W mutation in TBC1D1 has significant implications for protein function and metabolic regulation:

• Functional significance:

  • Disrupts the stable association between TBC1D1 and AMPK heterotrimers containing α1 subunits

  • Affects the kinetics of phosphorylation at Ser237, a critical AMPK-directed site

  • Associated with obesity predisposition and altered glucose metabolism in certain populations

  • May impair GLUT4 translocation and insulin-stimulated glucose uptake

• Experimental approaches to study R125W:

  • Site-directed mutagenesis to generate R125W mutant constructs

  • Stable cell lines expressing wild-type vs. R125W TBC1D1

  • CRISPR/Cas9 gene editing to introduce the mutation in relevant cell lines

  • Co-immunoprecipitation experiments to compare protein interactions

  • Phosphorylation kinetics studies comparing WT and R125W response to AMPK activators

• Analytical methods:

  • Use HRP-conjugated TBC1D1 antibody in Western blots to assess expression levels

  • Phospho-specific antibodies to monitor specific phosphorylation sites

  • Glucose uptake assays to assess functional impact

  • Live-cell imaging with fluorescently tagged GLUT4 to track translocation

• Data comparison table for WT vs. R125W TBC1D1:

This comprehensive approach provides insights into how this naturally occurring mutation affects TBC1D1 function and contributes to metabolic dysregulation.

How can researchers design experiments to investigate the differential roles of TBC1D1 in various muscle fiber types?

To investigate TBC1D1's differential roles across muscle fiber types:

• Experimental design considerations:

  • Select representative muscles: soleus (predominantly slow-twitch/oxidative), tibialis anterior or extensor digitorum longus (predominantly fast-twitch/glycolytic), and gastrocnemius (mixed fiber composition)

  • Compare TBC1D1 vs. AS160 expression and function across these muscle types

  • Design in vivo, ex vivo, and in vitro experiments to capture physiological relevance

• Methodological approaches:

  • Fiber type-specific isolation:

    • Laser capture microdissection of specific fiber types

    • Single fiber isolation from whole muscles

    • Primary culture of myoblasts from different muscle sources

  • Expression analysis:

    • Real-time PCR to quantify relative TBC1D1 and AS160 mRNA levels

    • Western blotting with HRP-conjugated TBC1D1 antibody to measure protein expression

    • Immunohistochemistry to visualize fiber type-specific distribution

  • Functional assessments:

    • Ex vivo contraction studies with muscle strips

    • Glucose uptake assays under basal, insulin, and AICAR-stimulated conditions

    • Phosphorylation analyses following various stimuli

• Advanced techniques:

  • Muscle-specific conditional knockout models

  • Fiber type-specific promoters for transgene expression

  • Metabolic flux analyses to assess substrate utilization

  • Proteomics to identify fiber type-specific TBC1D1 interactomes

• Translational considerations:

  • Exercise-induced adaptations in TBC1D1 expression and function

  • Fiber type shifts in metabolic diseases and their impact on TBC1D1 activity

  • Pharmacological targeting strategies based on fiber type-specific mechanisms

This comprehensive approach enables researchers to delineate the specialized roles of TBC1D1 across different muscle fiber types and their contributions to whole-body metabolism.

What are emerging techniques for studying the dynamics of TBC1D1 trafficking and interactions?

Emerging techniques for investigating TBC1D1 dynamics include:

• Advanced imaging approaches:

  • Live-cell super-resolution microscopy to track TBC1D1-containing vesicles

  • FRET/BRET biosensors to monitor real-time protein-protein interactions

  • Lattice light-sheet microscopy for 3D visualization of trafficking events

  • Correlative light and electron microscopy (CLEM) to link function with ultrastructure

• Proximity-based proteomics:

  • BioID or TurboID fusion proteins to identify proximal proteins in living cells

  • APEX2-based proximity labeling for temporal interaction mapping

  • Split-BioID systems to capture conditional interactions

• Single-cell analyses:

  • Single-cell proteomics to capture cell-to-cell variability in TBC1D1 function

  • Spatial transcriptomics to correlate TBC1D1 expression with cellular localization

  • Single-cell metabolomics to link TBC1D1 activity with metabolic outcomes

• Rapid kinetics methodologies:

  • Optogenetic control of TBC1D1 phosphorylation or localization

  • Microfluidic systems for precise temporal control of stimuli

  • Real-time monitoring of Rab GTPase activity using FRET-based sensors

These emerging technologies will provide unprecedented insights into the spatial and temporal dynamics of TBC1D1 function, offering new avenues for therapeutic targeting in metabolic disorders.

How might understanding TBC1D1 mechanisms contribute to developing novel therapeutic approaches for metabolic disorders?

Understanding TBC1D1 mechanisms offers several promising therapeutic avenues:

• Pathway-based interventions:

  • AMPK activators specifically targeting the TBC1D1-AMPK interaction

  • Small molecules enhancing TBC1D1 phosphorylation at Ser237

  • Compounds modulating the interaction between TBC1D1 and its trafficking partners (VPS13A, VPS13C)

• Tissue-specific approaches:

  • Skeletal muscle-targeted delivery systems leveraging the high expression of TBC1D1

  • Fast-twitch muscle fiber-specific interventions based on differential expression patterns

  • Exercise mimetics targeting TBC1D1-dependent pathways

• Personalized medicine applications:

  • Genetic screening for TBC1D1 variants (e.g., R125W) to identify at-risk individuals

  • Tailored interventions based on patient-specific TBC1D1 function

  • Pharmacogenomic approaches accounting for TBC1D1 polymorphisms

• Methodological considerations for drug development:

  • High-throughput screening assays using TBC1D1 GAP activity

  • Structure-based drug design targeting TBC1D1 functional domains

  • Phenotypic screening in relevant cellular systems with glucose uptake readouts

Advancing our understanding of TBC1D1 biology has significant potential to yield novel therapeutic strategies for disorders characterized by impaired glucose homeostasis, including type 2 diabetes and obesity.