TBK1 Antibody, Biotin conjugated

What is TBK1 and what cellular pathways does it regulate?

TBK1 (Tank-binding kinase 1) is a Ser/Thr kinase with a molecular weight of approximately 84 kDa that functions as a critical regulator in multiple cellular processes . TBK1 plays essential roles in:

• Innate immune responses and signaling

• Selective autophagy, particularly mitophagy (removal of damaged mitochondria)

• Cell cycle regulation

• Apoptotic pathways

• B cell differentiation and germinal center formation

TBK1 is particularly notable for its role in phosphorylating autophagy adaptors that mediate the selective autophagic removal of damaged mitochondria through PINK1-Parkin-mediated mitophagy . In B cells, TBK1 serves as a crucial determinant of germinal center commitment through fine-tuning of CD40 and BCR signaling pathways .

What are the advantages of using biotinylated TBK1 antibodies?

Biotinylated TBK1 antibodies offer several methodological advantages for research applications:

• Enhanced detection sensitivity through biotin-streptavidin amplification systems

• Versatility across multiple detection platforms (Western blotting, ELISA)

• Compatibility with various secondary detection systems

• Potential for multiplexed assays when combined with other primary antibodies

• Reduced background in many applications

• Ability to leverage streptavidin-conjugated reporter molecules for signal enhancement

Both monoclonal and polyclonal biotinylated TBK1 antibodies are available, offering researchers flexibility in experimental design based on specific requirements .

What are the key characteristics of commercially available TBK1 biotinylated antibodies?

Commercial TBK1 biotinylated antibodies have specific characteristics that researchers should consider:

These antibodies recognize TBK1 with high specificity and can detect both total and, depending on the epitope, potentially phosphorylated forms of the protein .

How should TBK1 biotinylated antibodies be used for Western blotting?

For optimal Western blotting results with TBK1 biotinylated antibodies:

• Sample preparation:

  • Extract proteins using lysis buffers containing protease and phosphatase inhibitors

  • Load 20-50 μg total protein per lane for cell/tissue lysates

• Electrophoresis and transfer:

  • Use 8-10% SDS-PAGE gels (appropriate for 84 kDa proteins)

  • Transfer to PVDF or nitrocellulose membrane at appropriate voltage

• Antibody incubation:

  • Block membrane with 5% non-fat milk or BSA in TBST

  • Dilute TBK1 biotinylated antibody 1:1000 in blocking solution

  • Incubate overnight at 4°C with gentle agitation

• Detection:

  • Incubate with streptavidin-HRP conjugate (1:2000-1:5000)

  • Develop using chemiluminescent substrate

Expected results: TBK1 protein should be detected at approximately 84 kDa, with potential slight mobility shifts if phosphorylated forms are present .

How can researchers optimize ELISA protocols using TBK1 biotinylated antibodies?

For optimizing ELISA assays with TBK1 biotinylated antibodies:

• Plate preparation:

  • Coat ELISA plates with capture antibody (anti-TBK1) at 1-5 μg/ml

  • Block plate thoroughly to minimize background

• Sample and antibody incubation:

  • Prepare samples in appropriate dilution buffer

  • Add biotinylated TBK1 antibody at optimized concentration

  • Maintain consistent incubation times and temperatures

• Detection optimization:

  • Use high-quality streptavidin-HRP conjugate

  • Optimize substrate incubation time for optimal signal-to-noise ratio

  • Include recombinant TBK1 protein as positive control

• Validation controls:

  • Include TBK1-deficient samples as negative controls

  • Test antibody specificity using competitive binding approaches

  • Verify linear range of detection

The polyclonal TBK1 antibody with biotin conjugation has been specifically validated for ELISA applications and shows strong reactivity with human TBK1 .

What species cross-reactivity should be expected with TBK1 biotinylated antibodies?

Species cross-reactivity depends on the specific TBK1 antibody:

• The TBK1/NAK (D1B4) Rabbit mAb (Biotinylated) demonstrates reactivity with human, mouse, rat, and monkey samples

• The rabbit polyclonal TBK1 biotinylated antibody has confirmed reactivity with human samples

When working with samples from different species, researchers should:

• Verify epitope conservation across species

• Perform preliminary validation experiments in the specific species of interest

• Include appropriate positive and negative controls from the target species

• Consider antibody concentrations may need adjustment for optimal detection in different species

Cross-reactivity data is particularly valuable for researchers conducting comparative studies across species or working with animal models of human disease .

How does TBK1 regulate B cell differentiation and germinal center formation?

TBK1 serves as a critical determinant in B cell differentiation and germinal center (GC) formation through several mechanisms:

• TBK1 activation during B cell differentiation:

  • TBK1 phosphorylation progressively increases during GC B cell differentiation

  • B cell-intrinsic TBK1 is absolutely required for GC formation

• Molecular regulation:

  • TBK1 modulates the balance between IRF4 and BCL6 expression

  • It achieves this by limiting CD40 and BCR activation through:

    • Noncanonical NF-κB signaling

    • AKT T308 phosphorylation pathways

• Consequences of TBK1 deficiency:

  • TBK1-deficient B cells can differentiate to Pre-GC stage but fail to form mature GCs

  • Without TBK1, CD40 and BCR signaling synergistically enhance IRF4 expression

  • Elevated IRF4 suppresses BCL6, preventing GC formation

  • Memory B cells generated from TBK1-deficient B cells fail to confer sterile immunity

Studies using B cell-specific TBK1-deficient mice demonstrate severely impaired GC formation in response to both immunization and infection models, despite normal T follicular helper cell differentiation .

How does TBK1 function in the OPTN-mediated mitophagy pathway?

TBK1 plays a sophisticated role in OPTN (optineurin)-mediated mitophagy through a positive feedback mechanism:

• Contact site formation:

  • OPTN serves as a platform for TBK1 activation

  • OPTN-ubiquitin and OPTN-PAS (pre-autophagosomal structure) interactions facilitate OPTN-TBK1 complex assembly

  • These complexes form at contact sites between damaged mitochondria and autophagosome formation sites

• Activation mechanism:

  • At the assembly point, TBK1 undergoes hetero-autophosphorylation at S172

  • This creates a positive feedback loop that accelerates further TBK1 activation

  • Activated TBK1 phosphorylates OPTN, enhancing its ubiquitin-binding capacity

• Expansion and downstream signaling:

  • TBK1 also phosphorylates RAB7A to promote ATG9A recruitment to damaged mitochondria

  • Additionally, TBK1 phosphorylates LC3C and GABARAPL2 to facilitate isolation membrane expansion

Experimental disruption of this pathway using engineered monobodies against OPTN impairs OPTN accumulation at contact sites, inhibits TBK1 activation, and consequently blocks mitochondrial degradation .

What experimental approaches can detect TBK1 activation in research models?

Several experimental approaches can effectively monitor TBK1 activation:

• Phosphorylation-specific detection:

  • Western blotting with phospho-specific antibodies targeting S172

  • Phospho-proteomics to identify TBK1 activation and substrate phosphorylation

  • Kinase activity assays using recombinant substrates

• Localization studies:

  • Immunofluorescence microscopy to visualize TBK1 recruitment to specific subcellular structures

  • Co-localization analysis with binding partners (OPTN, mitochondria, etc.)

  • Live-cell imaging with fluorescently tagged TBK1 to track activation dynamics

• Functional readouts:

  • Downstream substrate phosphorylation (OPTN, RAB7A, LC3C)

  • Formation of TBK1-dependent protein complexes

  • Phenotypic assays (mitophagy progression, germinal center formation)

When analyzing TBK1 activation, researchers should consider that some cell types exhibit basal TBK1 phosphorylation even under unstimulated conditions. To accurately measure activation-dependent phosphorylation, it may be necessary to subtract basal signals from post-stimulation measurements .

What are the key signaling mechanisms that regulate TBK1 activation?

TBK1 activation is regulated through several sophisticated mechanisms:

• Autophosphorylation:

  • Primary activation mechanism occurs through autophosphorylation at S172

  • This phosphorylation event is essential for converting TBK1 to its active form

  • Occurs during various stimuli including mitophagy and innate immune responses

• Adaptor protein interactions:

  • TBK1 directly interacts with OPTN and indirectly with NDP52 and TAX1BP1

  • These interactions facilitate TBK1 dimerization and clustering

  • Clustering increases local concentration, promoting trans-autophosphorylation

• Context-specific regulation:

  • In mitophagy: OPTN provides a platform for TBK1 activation at contact sites

  • In B cells: TBK1 phosphorylation increases during germinal center differentiation

  • In innate immunity: Pattern recognition receptor signaling activates TBK1

• Feedback mechanisms:

  • Positive feedback loops enhance TBK1 activation once initiated

  • Activated TBK1 phosphorylates binding partners, often creating feed-forward loops

Understanding these activation mechanisms is critical for interpreting experimental results and developing targeted interventions in TBK1-related pathways .

How can researchers troubleshoot inconsistent TBK1 detection in Western blots?

When experiencing inconsistent TBK1 detection in Western blots:

• Sample preparation issues:

  • Ensure complete protein extraction with appropriate lysis buffers

  • Include fresh protease and phosphatase inhibitors

  • Avoid repeated freeze-thaw cycles of samples

  • Confirm protein concentration determination is accurate

• Technical considerations:

  • Optimize antibody dilution (start with manufacturer's recommendation of 1:1000)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Ensure adequate blocking to reduce background

  • Verify transfer efficiency for high molecular weight proteins (84 kDa)

• Detection optimization:

  • Use freshly prepared ECL substrate

  • Adjust exposure time appropriately

  • Consider enhanced sensitivity detection systems for low abundance samples

  • Ensure streptavidin-HRP is functioning properly

• Controls and validation:

  • Include positive control samples known to express TBK1

  • Run TBK1-deficient samples as negative controls

  • Consider recombinant TBK1 protein as reference standard

Researchers should note that the recommended protocol specifically advises against aliquoting the antibody to maintain optimal performance .

What controls should be included when using TBK1 biotinylated antibodies?

Essential controls for experiments using TBK1 biotinylated antibodies include:

• Positive controls:

  • Cell lines known to express TBK1 endogenously

  • Samples treated with stimuli known to activate TBK1 (e.g., mitochondrial depolarizers)

  • Recombinant TBK1 protein (for calibration)

• Negative controls:

  • TBK1 knockout or knockdown samples when available

  • Isotype-matched biotinylated control antibody

  • Secondary detection reagent only (streptavidin without primary antibody)

• Specificity controls:

  • Comparison with non-biotinylated TBK1 antibody

  • Peptide competition assay

  • Verification of expected molecular weight (84 kDa)

• Technical controls:

  • Loading controls for Western blotting (housekeeping proteins)

  • Standard curves for quantitative applications

  • Replicate samples to assess reproducibility

Proper controls are essential for validating results and ensuring reliable interpretation of experimental data when working with biotinylated TBK1 antibodies.

How should researchers interpret unexpected TBK1 phosphorylation patterns?

When encountering unexpected TBK1 phosphorylation patterns:

• Basal phosphorylation considerations:

  • Some cell types exhibit constitutive TBK1 phosphorylation

  • Autophagy gene knockout cells can show elevated basal phospho-TBK1 levels

  • Calculate "newly generated" phospho-TBK1 by subtracting basal signals

• Context-dependent interpretation:

  • Consider the specific cellular pathway being studied

  • TBK1 participates in multiple pathways that may show different activation kinetics

  • Verify stimulus specificity and potential cross-pathway activation

• Temporal dynamics:

  • TBK1 phosphorylation may be transient or sustained depending on context

  • In mitophagy, phosphorylation increases progressively over hours

  • Establish proper time course experiments to capture activation dynamics

• Validation approaches:

  • Use TBK1 inhibitors to confirm specificity

  • Compare results with multiple phospho-specific antibodies

  • Correlate phosphorylation with downstream functional readouts

Research indicates that basal TBK1 phosphorylation should be considered when interpreting results, particularly in autophagy-deficient cells where phosphorylation signals may increase slowly during mitophagy induction .

What methodological considerations are important when studying TBK1 in different research models?

Key methodological considerations for TBK1 research across different models include:

• Cell/tissue-specific expression:

  • TBK1 functions differ between immune cells, neurons, and B cells

  • Antibody sensitivity may vary based on endogenous expression levels

  • Adjust protocols based on the specific biological context

• Species-specific considerations:

  • Verify antibody cross-reactivity with the species being studied

  • Consider epitope conservation across species

  • Select antibodies validated for specific applications in your species

• Activation context:

  • In B cells: Monitor during germinal center formation processes

  • In mitophagy: Examine after mitochondrial damage induction

  • In immune cells: Study following pathogen recognition receptor activation

• Technical adaptations:

  • Adjust lysis conditions based on subcellular localization

  • Consider cell-specific background signals

  • Optimize signal detection based on expression levels

• Model-specific controls:

  • Include tissue/cell-specific negative controls

  • Use genetically modified models when available

  • Consider pharmacological inhibitors for validation

These considerations ensure that experimental designs appropriately account for the biological context and technical requirements of different research models when studying TBK1 .