TBK1 (Ab-172) Antibody

What is TBK1 and why is phosphorylation at Serine 172 significant?

TBK1 is an 83.6 kDa serine/threonine protein kinase that functions as a critical signaling mediator in multiple cellular pathways. TBK1 activation occurs through trans-autophosphorylation at serine 172 (S172) in the activation loop within the kinase domain, making this modification the definitive marker of TBK1 activity . TBK1 serves dual roles in both promoting antiviral defenses and controlling TNF-mediated inflammation .

Phosphorylation at S172 is particularly significant because:

• It directly correlates with TBK1 enzymatic activity

• It serves as a biomarker for activated inflammatory and antiviral signaling pathways

• Elevated pS172-TBK1 levels have been detected in neurodegenerative conditions including Alzheimer's disease and frontotemporal dementia

• It indicates engagement of pattern recognition receptors including TLR3, RIG-I, MDA5, and cGAS/STING

Methodologically, researchers should note that baseline pS172-TBK1 levels vary by tissue type and disease state, necessitating appropriate controls when evaluating activation status.

What is the specific role of TBK1 in cellular signaling networks?

TBK1 functions as a signaling hub with multiple distinct roles:

• Antiviral signaling: TBK1 operates downstream of pattern recognition receptors (PRRs) including TLR3, RIG-I, MDA5, and cGAS/STING to activate interferon regulatory factors (IRFs) and trigger type I interferon (IFN-I) responses .

• Inflammatory regulation: TBK1 participates in NF-κB pathway activation but also negatively regulates TNF-mediated inflammatory cell death through inactivating interaction with RIPK1 .

• Autophagy regulation: TBK1 controls early stages of autophagy, with important implications for cellular homeostasis .

• Tau modification: TBK1 acts as a tau kinase that can directly phosphorylate tau protein, potentially contributing to tau hyperphosphorylation in tauopathies .

When designing experiments, researchers should consider which specific TBK1 function they are investigating and select appropriate readouts beyond mere activation status.

What experimental systems best demonstrate TBK1 activation dynamics?

The most robust experimental systems for studying TBK1 activation include:

• Cell culture models with pattern recognition receptor stimulation:

  • Treatment with poly(I:C) for TLR3 or RIG-I pathway activation

  • Cytosolic DNA transfection for cGAS/STING activation

  • These stimuli consistently induce detectable TBK1 phosphorylation within 30-60 minutes

• TNF-induced cell death models:

  • Cell lines sensitized to necroptosis (e.g., with caspase inhibitors) show prominent TBK1 activation

  • Patient-derived fibroblasts from TBK1-deficient individuals exhibit increased sensitivity to TNF-induced cell death

• Neurodegenerative disease models:

  • Tau transgenic mouse models show progressive increase in pS172-TBK1 levels correlating with disease progression

  • Human AD and FTDP-17 brain tissues demonstrate significantly increased TBK1 activation compared to controls

Methodologically, it is crucial to verify TBK1 activation using both phospho-specific antibodies and functional readouts (e.g., downstream IRF3 phosphorylation or interferon-stimulated gene expression).

What are the optimal protocols for detecting phosphorylated TBK1 using antibody-based methods?

When using TBK1 (Ab-172) antibody for detecting phosphorylated TBK1, the following optimized protocols yield reliable results:

For Western Blotting:

• Sample preparation: Lyse cells in buffer containing phosphatase inhibitors (e.g., 25 mM MOPS, pH 7.2, 12.5 mM β-glycerol-phosphate, 25 mM MgCl₂, 5 mM EGTA, 2 mM EDTA and 0.25 mM DTT)

• Protein quantification: Bradford or BCA assay

• Sample loading: 20-40 μg total protein per lane

• Resolution: 8% SDS-PAGE gels provide optimal separation for the 83.6 kDa TBK1 protein

• Transfer: Semi-dry transfer at 15V for 30 minutes or wet transfer at 100V for 1 hour

• Blocking: 5% BSA in TBST (superior to milk which contains phosphatases)

• Primary antibody: TBK1 (Ab-172) at 1:1000 dilution, overnight at 4°C

• Detection: HRP-conjugated secondary antibody with ECL detection system

For Immunohistochemistry/Immunofluorescence:

• Fixation: 4% paraformaldehyde for 15 minutes

• Permeabilization: 0.1% Triton X-100 for 10 minutes

• Blocking: 10% serum from secondary antibody host species for 1 hour

• Primary antibody: TBK1 (Ab-172) at 1:200 dilution, overnight at 4°C

• Secondary detection: Fluorophore-conjugated secondary antibody (1:500) or HRP-conjugated polymer system

Always include appropriate positive controls (e.g., poly(I:C)-stimulated cells) and negative controls (e.g., lambda phosphatase-treated samples) to validate phospho-specificity.

How can researchers design experiments to study TBK1's dual roles in immunity and neurodegeneration?

To effectively study TBK1's functions across different biological contexts, consider these experimental design approaches:

For Immune Function Studies:

• Cell models:

  • Primary human fibroblasts (normal vs. TBK1-deficient)

  • Macrophages with TBK1 knockdown/knockout

  • Reconstitution experiments with wild-type or kinase-dead TBK1

• Stimulation paradigms:

  • Endosomal poly(I:C) (TLR3-dependent)

  • Cytoplasmic poly(I:C) (RIG-I/MDA5-dependent)

  • TNF with/without caspase inhibitors (cell death pathways)

• Readouts:

  • pS172-TBK1 levels via Western blot

  • IFN-β and IL-6 production (ELISA or qRT-PCR)

  • Cell death quantification (e.g., propidium iodide/annexin V staining)

For Neurodegenerative Disease Studies:

• Models:

  • Human postmortem brain tissue (AD, FTDP-17)

  • Primary neurons with tau pathology

  • TBK1 inhibitor treatment paradigms

• Analytical approaches:

  • Co-immunoprecipitation of TBK1 with tau

  • Immunohistochemistry for pS172-TBK1 colocalization with tau aggregates

  • In vitro kinase assays using recombinant TBK1 and tau

• Data validation:

  • TBK1 inhibitors (e.g., BX795) to confirm kinase-dependent effects

  • Phospho-site mapping via mass spectrometry

Crucially, researchers should design experiments that directly compare TBK1's functions across different cellular contexts to understand context-dependent regulation and function.

What are the critical considerations for in vitro kinase assays with TBK1?

When conducting in vitro kinase assays to assess TBK1 activity toward substrates like tau, researchers should implement the following methodology:

Standard In Vitro Kinase Assay Protocol:

• Reaction components:

  • 0.5 μg active TBK1 (e.g., recombinant protein from baculovirus expression system)

  • 1 μg substrate protein (e.g., full-length tau)

  • Kinase buffer: 25 mM MOPS (pH 7.2), 12.5 mM β-glycerol-phosphate, 25 mM MgCl₂, 5 mM EGTA, 2 mM EDTA, 0.25 mM DTT

  • 100 μM ATP

  • Optional: [γ-³²P]ATP for radioactive detection

• Reaction conditions:

  • 30 minutes at 37°C

  • For time course analysis: Sample aliquots at 0, 10, 20, 30, 60 minutes

  • For inhibitor studies: Pre-incubate TBK1 with inhibitor (e.g., BX795 at 5-40 μM) before adding substrate

• Analysis methods:

  • Western blot with phospho-specific antibodies

  • Mass spectrometry for comprehensive phosphosite mapping

  • SDS-PAGE with autoradiography (if using radioactive ATP)

Critical Control Experiments:

• Kinase-dead TBK1 control (K38A mutant)

• ATP omission control

• Inhibitor dose-response curves (BX795 or MRT67307)

• Substrate specificity controls (various tau constructs or deletion mutants)

This approach enables rigorous identification of direct TBK1 substrates and phosphorylation sites.

How can researchers leverage TBK1 (Ab-172) antibody to study the connections between autophagy and innate immunity?

TBK1 functions as a crucial bridge between autophagy and innate immunity. To investigate this connection:

Experimental Approach 1: Selective Autophagy of Pathogens

• Infect cells with fluorescently-labeled bacteria or transfect with viral components

• Perform time-course immunofluorescence with TBK1 (Ab-172) antibody and autophagy markers (LC3, p62/SQSTM1)

• Quantify colocalization of phosphorylated TBK1 with autophagy receptors and pathogen components

• Compare wild-type cells with autophagy-deficient cells (ATG5 KO, ATG7 KO)

Experimental Approach 2: Activation-Dependent TBK1 Complexes

• Stimulate cells with specific PRR ligands (cGAMP, poly(I:C), etc.)

• Perform immunoprecipitation with TBK1 (Ab-172) antibody

• Analyze precipitated complexes via mass spectrometry or immunoblotting for autophagy adaptors

• Validate interactions with proximity ligation assays

Key Research Questions:

• Does selective autophagy require TBK1 kinase activity?

• Which autophagy adaptors are phosphorylated by TBK1 under different stimulation conditions?

• How do disease-associated TBK1 variants affect both autophagy and interferon responses?

This integrated approach allows researchers to dissect the dual roles of TBK1 in coordinating antimicrobial autophagy and interferon production.

What methodologies best elucidate TBK1's role in tau pathology and neurodegeneration?

To investigate TBK1's contribution to tauopathies like Alzheimer's disease and FTDP-17:

Analytical Framework for Tau-TBK1 Interactions:

• Co-immunoprecipitation studies:

  • Immunoprecipitate tau from postmortem brain tissues (AD, FTDP-17, controls)

  • Blot for TBK1 and phospho-TBK1

  • Quantify relative interaction in disease vs. control samples

• Mass spectrometry phosphosite mapping:

  • Perform in vitro kinase assays with recombinant TBK1 and tau

  • Digest with trypsin

  • Analyze peptides using LC-MS/MS with phospho-enrichment

  • Map identified phosphosites to tau structure and pathology-associated sites

• Functional validation in neuronal models:

  • Transfect neurons with tau and wild-type or mutant TBK1

  • Treat with TBK1 inhibitors at various doses (5-40 μM)

  • Assess tau phosphorylation, aggregation, and neurotoxicity

  • Quantify neuronal health using viability assays, neurite outgrowth, or electrophysiology

Data Analysis Matrix for Tau-TBK1 Studies:

This comprehensive approach allows researchers to establish both correlation and causation in TBK1's contribution to tau pathology.

How does TBK1 activation influence regulated cell death pathways, and what methodological approaches are optimal for this investigation?

TBK1 plays a critical role in regulating various forms of cell death, particularly TNF-induced cell death. To investigate this:

Methodological Approach:

• Cell death induction protocols:

  • TNF (10-50 ng/mL) alone or with sensitizers:

    • Smac mimetics (inhibit cIAPs)

    • Caspase inhibitors (z-VAD-fmk to shift toward necroptosis)

    • Cycloheximide (protein synthesis inhibitor)

  • Time-course analysis: 2, 4, 8, 16, 24 hours

• Cell death analysis methods:

  • Flow cytometry (Annexin V/propidium iodide)

  • Fluorescent plate reader assays (calcein-AM/ethidium homodimer)

  • Immunoblotting for cleaved caspase-3 (apoptosis) or phospho-MLKL (necroptosis)

  • LDH release assays for membrane permeabilization

• Signaling analysis:

  • Immunoprecipitate RIPK1 complexes

  • Blot for TBK1, phospho-TBK1, and complex components

  • Compare formation of complex I (signaling) vs. complex II (death-inducing)

Validation using genetic models:

• TBK1-deficient patient fibroblasts show increased necroptosis in response to TNF

• This phenotype can be rescued with anti-TNF treatment

• Pharmacological inhibition with TBK1 inhibitors should phenocopy genetic deficiency

This methodological framework enables comprehensive characterization of TBK1's regulatory role in cell death decisions.

What are common technical issues with phospho-TBK1 detection and their solutions?

Researchers frequently encounter these challenges when detecting phosphorylated TBK1:

Methodological Solution for Phospho-Stability:
For challenging samples like brain tissue, implement this optimized protocol:

• Rapid dissection and flash-freezing in liquid nitrogen

• Homogenization in buffer containing 1% SDS, 1 mM EDTA, 5 mM NaF, 2 mM Na₃VO₄, 10 mM β-glycerophosphate

• Immediate heat-denaturation (95°C for 5 minutes)

• Dilution in non-denaturing buffer for immunoprecipitation if needed

This approach maximally preserves phosphorylation status for accurate assessment.

How should researchers address contradictory results between TBK1 phosphorylation and downstream effector activation?

When TBK1 phosphorylation states do not align with expected downstream effects, consider this analytical framework:

Systematic Troubleshooting Approach:

• Pathway component analysis:

  • Check expression levels of all pathway components (e.g., IRF3, STING, RIPK1)

  • Assess phosphorylation of multiple sites on TBK1 beyond S172

  • Evaluate adapter molecule availability and complex formation

• Temporal dynamics:

  • Perform detailed time-course analysis (TBK1 activation often precedes downstream effects)

  • Consider transient vs. sustained activation patterns

  • Assess negative regulatory mechanisms (phosphatases, feedback inhibitors)

• Context-dependent signaling:

  • TBK1 functions differently in IFN-I induction vs. autophagy vs. cell death regulation

  • In TBK1-deficient patients, NF-κB-dependent IL-6 induction remains nearly intact while IFN-I induction is hypomorphic

  • The system exhibits plasticity with compensatory mechanisms (e.g., IKKε)

• Validation strategies:

  • Complementation experiments with wild-type vs. mutant TBK1

  • Chemical inhibition with different TBK1 inhibitors (BX795, MRT67307)

  • Genetic epistasis experiments placing TBK1 in the pathway context

This structured approach helps reconcile apparently contradictory observations by understanding the complex regulatory networks controlling TBK1 function.

What controls are essential for validating TBK1 (Ab-172) antibody specificity?

To ensure reliable and specific detection of phosphorylated TBK1, implement these critical controls:

Essential Experimental Controls:

• Genetic controls:

  • TBK1 knockout or knockdown cells (negative control)

  • Cells expressing TBK1 S172A mutant (phospho-site mutant)

  • Rescue with wild-type vs. kinase-dead TBK1

• Treatment controls:

  • Positive control: Poly(I:C) stimulation (6-8 hours) to induce robust TBK1 phosphorylation

  • Negative control: Unstimulated cells with minimal basal phosphorylation

  • Inhibitor control: Pre-treatment with TBK1 inhibitors (BX795 at 5-40 μM)

• Technical validation:

  • Phosphatase treatment: Sample aliquot treated with lambda phosphatase to demonstrate phospho-specificity

  • Peptide competition: Pre-incubation of antibody with phospho-peptide vs. non-phospho-peptide

  • Multiple antibody validation: Confirm results with independent phospho-TBK1 antibodies

• Functional correlation:

  • Parallel assessment of downstream substrates (IRF3 phosphorylation)

  • Functional readouts (IFN-β induction, reporter assays)

  • Kinase activity assays with immunoprecipitated TBK1

Implementing these comprehensive controls ensures that observed signals genuinely reflect TBK1 phosphorylation status rather than artifacts.

How can TBK1 (Ab-172) antibody contribute to understanding the therapeutic potential of TBK1 inhibition?

TBK1 inhibition represents a promising therapeutic strategy for multiple conditions. The TBK1 (Ab-172) antibody provides a critical tool for evaluating inhibitor efficacy and mechanism:

Therapeutic Target Validation Framework:

• In vitro inhibitor characterization:

  • BX795 and derivatives (MRT67307) show dose-dependent inhibition of TBK1 (IC₅₀ values in nanomolar range)

  • Western blotting with TBK1 (Ab-172) antibody provides direct measure of target engagement

  • In vitro kinase assays with tau or other substrates demonstrate functional inhibition

• Cellular model testing:

  • Patient-derived cells from TBK1-deficient individuals serve as "natural knockout" models

  • Inhibitor treatment should phenocopy genetic deficiency

  • Anti-TNF treatment rescues inflammatory phenotypes in TBK1-deficient models

• Disease-specific applications:

  • Tauopathies: TBK1 inhibitors prevent tau hyperphosphorylation by TBK1

  • Autoinflammatory conditions: Inhibition reduces TNF-induced cell death

  • Combination therapies: TBK1 inhibitors with anti-TNF biologics

Biomarker Development Strategy:

• TBK1 (Ab-172) antibody can monitor treatment efficacy in clinical samples

• Phospho-TBK1 levels serve as pharmacodynamic biomarkers

• Correlation between pTBK1 reduction and clinical improvement validates mechanism

This approach enables rational development of TBK1-targeted therapeutics for multiple disease indications.

What novel methodologies are emerging for studying TBK1 phosphorylation dynamics in real-time?

Recent technological advances offer new opportunities to study TBK1 activation dynamics with unprecedented temporal and spatial resolution:

Emerging Methodological Approaches:

• Biosensor development:

  • FRET-based TBK1 activity sensors using phospho-binding domains

  • Split luciferase complementation systems for S172 phosphorylation

  • These tools enable live-cell imaging of TBK1 activation

• Advanced microscopy techniques:

  • Super-resolution microscopy of TBK1 signaling complexes

  • Lattice light-sheet microscopy for 3D visualization of TBK1 translocation

  • Single-molecule tracking of TBK1 during activation

• Mass spectrometry innovations:

  • Targeted proteomics assays for quantitative measurement of TBK1 phosphorylation

  • Phospho-proteomic analysis of TBK1 substrates using TMT labeling

  • Kinetic isotope labeling to track temporal dynamics of phosphorylation

• Multiplexed detection systems:

  • Automated high-content imaging with machine learning analysis

  • Simultaneous detection of multiple phosphorylation events

  • Correlation of TBK1 activation with cellular phenotypes

These technologies will enable researchers to address fundamental questions about the spatiotemporal regulation of TBK1 activity in different cellular compartments and disease contexts.

How might researchers investigate the intersection of TBK1 function in both neurodegeneration and immune regulation?

The dual role of TBK1 in neurodegeneration and immunity presents a unique opportunity for interdisciplinary research:

Integrated Research Framework:

• Comparative analysis across tissues:

  • Parallel analysis of TBK1 activation in immune cells and neurons

  • Cross-tissue validation in patient samples (e.g., CSF vs. PBMCs)

  • Single-cell analysis to identify cell type-specific activation patterns

• Disease-relevant model systems:

  • Human iPSC-derived microglia and neurons from patients with TBK1 mutations

  • 3D brain organoids with integrated immune components

  • Humanized mouse models with tissue-specific TBK1 modulation

• Multi-modal assessment approaches:

  • Correlate pTBK1 levels with inflammatory markers and neurodegeneration

  • Relate neurodegenerative phenotypes to immune dysregulation

  • In AD and FTDP-17 patients, increased TBK1 activation correlates with tau pathology

• Translational applications:

  • Early TBK1 activation as a biomarker for neuroinflammation

  • Targeted inhibition of TBK1 in specific cellular compartments

  • Combined targeting of tau phosphorylation and inflammatory cascades

This integrated approach may reveal how TBK1 functions as a molecular link between neuroinflammation and neurodegeneration, potentially identifying novel therapeutic strategies that address both processes simultaneously.