TBK1 Antibody, FITC conjugated

What is TBK1 and why is it significant in immunological research?

TBK1 (TANK-binding kinase 1) is a serine/threonine protein kinase that plays essential roles in regulating inflammatory responses to foreign agents. It functions as a key mediator in innate immune signaling pathways, particularly for type I interferon (IFN) production following nucleic acid recognition. TBK1 associates with TRAF3 and TANK upon activation of toll-like receptors, subsequently phosphorylating interferon regulatory factors (IRFs) IRF3 and IRF7 as well as DDX3X . This phosphorylation allows homodimerization and nuclear translocation of these factors, leading to transcriptional activation of pro-inflammatory and antiviral genes including IFNA and IFNB. Beyond innate immunity, TBK1 has recently been identified as a crucial B cell-intrinsic factor for germinal center formation, highlighting its unexpected role in adaptive immunity . These multifaceted functions make TBK1 a significant target for immunological research.

What specific applications are FITC-conjugated TBK1 antibodies optimized for?

FITC-conjugated TBK1 antibodies are specifically optimized for fluorescence-based detection methods including:

• Immunofluorescence (IF) - For visualizing subcellular localization of TBK1 in fixed cells and tissues

• Flow cytometry (FC) - For quantitative analysis of TBK1 expression in cell populations

• Enzyme-linked immunosorbent assay (ELISA) - For quantitative detection of TBK1 in solution

• Immunohistochemistry with paraffin-embedded sections (IHC-P) - For detection of TBK1 in tissue specimens

These antibodies can detect TBK1 protein from multiple species including human, mouse, and rat origin, making them versatile tools for comparative immunological studies . The FITC (fluorescein isothiocyanate) conjugation eliminates the need for secondary antibody incubation steps, simplifying protocols and reducing background in fluorescence-based applications.

What methods should be used for validating TBK1 antibody specificity?

Validating TBK1 antibody specificity is critical for experimental reliability. Recommended validation methods include:

• Western blot analysis comparing TBK1-expressing and TBK1-knockout/knockdown samples

• Peptide competition assays using the immunizing peptide sequence from Serine/threonine-protein kinase TBK1 protein (644-661AA)

• Immunoprecipitation followed by mass spectrometry to confirm target identity

• Cross-validation using multiple antibodies targeting different epitopes of TBK1

• Testing for cross-reactivity with related kinases like IKKε

For FITC-conjugated antibodies specifically, validation should include fluorescence minus one (FMO) controls for flow cytometry applications and confirmation of expected subcellular localization patterns in immunofluorescence. The antibody should recognize the expected ~84 kDa band in western blots when using cell lysates known to express TBK1 .

What are the optimal sample preparation methods for TBK1 detection using FITC-conjugated antibodies?

Optimal sample preparation for TBK1 detection varies by application but should follow these general principles:

For Flow Cytometry:

• Harvest cells and wash in PBS containing 1-2% BSA

• Fix cells with 4% paraformaldehyde for 10-15 minutes at room temperature

• Permeabilize with 0.1-0.5% Triton X-100 or saponin buffer for intracellular staining

• Block with 5-10% normal serum from the same species as the secondary antibody

• Incubate with FITC-conjugated TBK1 antibody at recommended dilution (typically 1:50-1:200)

• Wash thoroughly to remove unbound antibody before analysis

For Immunofluorescence:

• Fix cells or tissue sections with 4% paraformaldehyde

• Permeabilize with 0.1-0.5% Triton X-100

• Block with 5-10% normal serum

• Incubate with FITC-conjugated TBK1 antibody overnight at 4°C

• Counterstain nuclei with DAPI

• Mount using anti-fade mounting medium to preserve FITC fluorescence

The critical factor for both applications is effective permeabilization, as TBK1 is primarily localized in the cytoplasm with translocation to specific subcellular compartments upon activation.

How can researchers overcome high background when using FITC-conjugated TBK1 antibodies?

High background is a common challenge when using FITC-conjugated antibodies. Effective strategies include:

• Optimize antibody concentration: Titrate the antibody to determine the optimal concentration that maximizes specific signal while minimizing background. Recommended starting dilutions are 1:50-1:200

• Improve blocking: Use 5-10% normal serum from the species in which the primary antibody was raised (rabbit for polyclonal or mouse for monoclonal) combined with 1% BSA to reduce non-specific binding

• Reduce autofluorescence:

  • For fixed tissues: Treat with 0.1-1% sodium borohydride solution for 10 minutes

  • For cells with high flavoprotein content: Pre-treatment with 10mM CuSO₄ in 50mM ammonium acetate buffer (pH 5.0)

  • Consider switching to longer wavelength fluorophores if FITC channel autofluorescence persists

• Increase washing stringency: Use PBS-T (PBS + 0.05-0.1% Tween-20) and extend washing times between incubation steps

• Optimize fixation: Overfixation can increase autofluorescence. Test different fixation durations and concentrations

• Use appropriate negative controls: Include isotype controls and secondary-only controls to assess non-specific binding

What controls should be included when using FITC-conjugated TBK1 antibodies?

A robust experimental design using FITC-conjugated TBK1 antibodies should include the following controls:

Essential Controls:

• Isotype control: A FITC-conjugated non-specific antibody of the same isotype (IgG1 κ for monoclonal or rabbit IgG for polyclonal) to assess non-specific binding

• Negative biological control: Samples known to express minimal TBK1 or TBK1-knockout samples

• Positive biological control: Samples with confirmed TBK1 expression or overexpression

• Blocking peptide control: Pre-incubation of the antibody with the immunizing peptide to confirm specificity

Application-Specific Controls:

• For flow cytometry: Unstained cells, single-stained controls for each fluorophore, and FMO (Fluorescence Minus One) controls

• For immunofluorescence: Secondary-only control and autofluorescence control (fixed cells without any antibody)

• For experimental treatments: Include appropriate vehicle controls for any treatments that may affect TBK1 expression or activation

Documenting these controls thoroughly is essential for publication and reproducibility of findings.

How can FITC-conjugated TBK1 antibodies be used to investigate TBK1's role in B cell function and germinal center formation?

Recent research has identified TBK1 as a crucial B cell-intrinsic factor for germinal center formation . FITC-conjugated TBK1 antibodies offer several methodological approaches to investigate this role:

• Flow cytometric analysis of germinal center B cells:

  • Use multicolor flow cytometry combining FITC-conjugated TBK1 antibodies with markers for germinal center B cells (CD19+CD95+GL7+)

  • Quantify TBK1 expression levels at different stages of B cell differentiation (naive, activated, pre-GC, GC, plasma cells, and memory B cells)

  • Correlate TBK1 expression with key transcription factors like BCL6 and IRF4

• Immunofluorescence microscopy of lymphoid tissues:

  • Perform IF staining of lymphoid tissue sections to visualize TBK1 expression within germinal center architecture

  • Co-stain with markers for follicular dendritic cells, T follicular helper cells, and B cell subsets

  • Track TBK1 phosphorylation status during germinal center reactions

• In vitro B cell differentiation assays:

  • Monitor TBK1 phosphorylation during in vitro activation of B cells with CD40L and IL-21 to mimic T cell help

  • Track correlations between TBK1 activity and expression of key B cell differentiation markers

  • Assess the impact of TBK1 inhibitors on germinal center B cell differentiation

These approaches can help elucidate how TBK1 regulates the balance of IRF4/BCL6 expression by modulating CD40 and BCR signaling through noncanonical NF-κB and AKT signaling pathways .

What methods can detect both TBK1 protein localization and its activation status simultaneously?

Detecting both TBK1 localization and activation status simultaneously requires sophisticated immunofluorescence approaches:

• Dual immunofluorescence with phospho-specific antibodies:

  • Combine FITC-conjugated TBK1 antibody (for total protein) with a different fluorophore-conjugated phospho-TBK1 antibody

  • This allows visualization of both total TBK1 distribution and its activated (phosphorylated) form

  • Calculate the phospho-to-total TBK1 ratio as a measure of activation across different subcellular compartments

• Proximity ligation assay (PLA):

  • Use PLA to detect TBK1 in close proximity to its binding partners or substrates

  • This approach can reveal activation-dependent interactions with proteins like TANK, IRF3, or AKT

  • Quantify PLA signals as indicators of functional TBK1 complexes

• Live-cell imaging with biosensors:

  • For dynamic studies, develop FRET-based biosensors incorporating TBK1 substrate domains

  • This allows real-time visualization of TBK1 kinase activity in living cells

  • Correlate kinase activity with subcellular redistribution during cell activation

These techniques provide complementary information about where TBK1 is located and whether it is enzymatically active in those locations, offering deeper insights into its function in various cellular contexts.

How can researchers investigate the TBK1-AKT pathway interaction using FITC-conjugated TBK1 antibodies?

The interaction between TBK1 and AKT represents an important signaling node in cellular survival pathways . FITC-conjugated TBK1 antibodies can be employed in several approaches to study this pathway:

• Co-immunoprecipitation and proximity assays:

  • Use FITC-conjugated TBK1 antibodies in fluorescence-based co-IP workflows

  • Perform proximity ligation assays (PLA) between TBK1 and AKT to visualize their direct interaction in situ

  • The data indicates that TBK1 and AKT associate through their respective kinase domains

• Kinase activity assays:

  • Develop fluorescence-based kinase assays using FITC-TBK1 antibodies to pull down the kinase

  • Measure phosphorylation of known substrates like GSK3α/β fusion peptide as described in previous studies

  • Compare kinase activity in the presence of PI3K inhibitors to assess pathway independence

• Phosphorylation site mapping:

  • Combine immunofluorescence with phospho-specific antibodies against AKT T308 and S473

  • Correlate TBK1 expression/activity with AKT phosphorylation status

  • Previous research indicates TBK1 can induce AKT activation independently of the canonical PDK1/mTORC2 pathway

• Inhibitor studies:

  • Treat cells with TBK1-specific inhibitors (like Compound II) and monitor effects on AKT pathway activation

  • Document changes in AKT substrate phosphorylation and compare with PI3K inhibitors like LY294002

  • Research suggests TBK1 inhibition blocks AKT pathway activation at doses comparable to those affecting IRF-3 nuclear localization

This table summarizes comparative effects of pathway inhibition based on previous research findings .

What factors affect the sensitivity and specificity of FITC-conjugated TBK1 antibody detection?

Several factors can significantly impact the performance of FITC-conjugated TBK1 antibodies:

• Antibody quality and origin:

  • Polyclonal antibodies provide broader epitope recognition but may have batch-to-batch variability

  • Monoclonal antibodies offer consistent specificity but may be sensitive to epitope masking

  • Recombinant monoclonal antibodies typically provide superior consistency

• FITC conjugation ratio:

  • Over-labeling can cause fluorophore quenching and reduced sensitivity

  • Under-labeling results in weak signal

  • Optimal fluorophore-to-protein ratio should be verified for each lot

• Sample preparation factors:

  • Fixation method and duration affect epitope preservation

  • Permeabilization efficiency influences antibody access to intracellular TBK1

  • Blocking effectiveness impacts background signal

  • For phospho-TBK1 detection, phosphatase inhibitors are essential during sample preparation

• Technical considerations:

  • FITC photobleaching during extended imaging sessions

  • pH sensitivity of FITC (optimal at pH 7.4-8.0)

  • Spectral overlap with other fluorophores in multiplex experiments

  • Instrumentation settings (voltage, compensation, gain)

• Biological factors:

  • Expression level of TBK1 in target cells

  • Activation state affecting epitope accessibility

  • Protein-protein interactions potentially masking antibody binding sites

  • Post-translational modifications affecting antibody recognition

Researchers should systematically evaluate these factors when troubleshooting or optimizing TBK1 detection protocols.

How can researchers design experiments to study TBK1's dual role in innate and adaptive immunity?

TBK1's involvement in both innate immunity (via type I IFN production) and adaptive immunity (via B cell functions) requires carefully designed experiments to dissect these dual roles. Recommended approaches include:

• Cell-specific conditional knockout models:

  • Compare phenotypes between myeloid-specific, B cell-specific, and T cell-specific TBK1 knockout mice

  • Challenge with pathogens or immunization protocols that engage both innate and adaptive immunity

  • The TBK1-deficient B cell model revealed that TBK1-deficient B cells failed to form germinal centers despite normal T follicular helper cell differentiation

• Temporal inhibition studies:

  • Use inducible knockout systems or timed inhibitor treatment to distinguish between TBK1's early (innate) and late (adaptive) functions

  • Monitor both interferon production and germinal center formation in the same experimental system

• Pathway-specific readouts:

  • Measure IRF3/7 phosphorylation and interferon production for innate functions

  • Assess BCL6/IRF4 balance and germinal center formation for adaptive functions

  • Previous research shows TBK1 phosphorylation regulates the balance of IRF4/BCL6 expression by limiting CD40 and BCR activation

• Protein interaction mapping:

  • Compare TBK1 protein complexes in innate immune cells versus B cells

  • Identify cell type-specific interaction partners that may direct TBK1 toward different downstream pathways

  • Research indicates TBK1 forms different complexes depending on cell type and cellular stimuli

These experimental approaches can help delineate the mechanistic basis for TBK1's context-specific functions across the immune system.

What are the critical considerations when using FITC-conjugated TBK1 antibodies in multiplex immunofluorescence assays?

Multiplex immunofluorescence involving FITC-conjugated TBK1 antibodies requires special considerations:

• Spectral overlap management:

  • FITC emission spectrum overlaps with other green fluorophores like GFP and Alexa Fluor 488

  • Careful panel design with sufficient spectral separation between fluorophores

  • Proper compensation controls for flow cytometry or spectral unmixing for microscopy

• Antibody panel optimization:

  • Prioritize brighter fluorophores for lower abundance targets

  • Consider placing TBK1-FITC on a detector with high sensitivity if TBK1 expression is low

  • Test for antibody cross-reactivity and fluorophore interactions

• Sequential staining approach:

  • For difficult combinations, employ sequential staining with intermediate fixation steps

  • This can help overcome issues with antibody cross-reactivity or steric hindrance

  • May be necessary when combining TBK1 detection with its binding partners or substrates

• Quantitative accuracy:

  • Include single-stained controls for each fluorophore

  • Add fluorescence minus one (FMO) controls

  • Use spectral viewers and panel design tools to predict and mitigate fluorescence spillover

• Image acquisition settings:

  • FITC is susceptible to photobleaching, so minimize exposure time

  • Acquire FITC channel early in the imaging sequence

  • Consider anti-fade mounting media specifically optimized for FITC preservation

By addressing these considerations, researchers can successfully incorporate FITC-conjugated TBK1 antibodies into multiplex assays to study TBK1 in complex cellular contexts.

How can FITC-conjugated TBK1 antibodies contribute to understanding TBK1's role in cancer?

TBK1 has emerging significance in cancer biology through its roles in survival signaling, particularly via the AKT pathway . FITC-conjugated TBK1 antibodies enable several research approaches:

• Profiling TBK1 expression across cancer types:

  • Use flow cytometry with FITC-conjugated TBK1 antibodies to quantify expression in patient-derived samples

  • Correlate expression levels with clinical outcomes and treatment responses

  • Research indicates TBK1 inhibition affects AKT pathway activation and survival in multiple cancer cell lines

• Spatial profiling in tumor microenvironment:

  • Apply multiplex immunofluorescence to map TBK1 expression and activation in different cell populations within tumors

  • Correlate with immune infiltration patterns and checkpoint molecule expression

  • Assess relationship between TBK1 activity and immunosuppressive features

• Therapeutic response monitoring:

  • Track changes in TBK1 expression/phosphorylation following treatment with targeted therapies

  • Assess whether TBK1 activation serves as a resistance mechanism to PI3K/AKT inhibitors

  • Previous work shows TBK1 expression can drive AKT activation despite PI3K family inhibition

• Functional studies in cancer models:

  • Combine FITC-conjugated TBK1 antibody staining with functional readouts of cancer cell behavior

  • Correlate TBK1 activity with proliferation, migration, and therapy resistance phenotypes

  • Determine how TBK1 inhibition compares with established PI3K/AKT pathway inhibitors

These approaches could help determine whether TBK1 represents a viable therapeutic target in specific cancer contexts and identify biomarkers for patient stratification.

What methodological approaches can assess the impact of TBK1 on vaccine responses?

Recent findings on TBK1's role in germinal center formation have significant implications for vaccine development . Researchers can use FITC-conjugated TBK1 antibodies in these methodological approaches:

• Tracking TBK1 dynamics post-vaccination:

  • Monitor TBK1 expression and phosphorylation in B cells at various timepoints after vaccination

  • Compare primary versus recall responses to assess memory B cell involvement

  • Research indicates memory B cells generated from TBK1-deficient B cells fail to confer sterile immunity upon reinfection

• Correlative studies with antibody quality:

  • Assess relationship between B cell TBK1 activity and subsequent antibody affinity maturation

  • Track somatic hypermutation rates in relation to TBK1 expression levels

  • Measure long-term antibody persistence in models with varying TBK1 function

• Adjuvant response studies:

  • Evaluate how different vaccine adjuvants affect TBK1 activation in B cells

  • Determine whether adjuvants targeting TBK1 pathways enhance germinal center responses

  • Test whether TBK1 enhancement can improve responses in immunocompromised models

• Vaccination protocols in TBK1-modified models:

  • Compare standard vaccination protocols in wild-type versus B cell-specific TBK1-deficient models

  • Test whether modified vaccination schedules can overcome TBK1 deficiency

  • Assess the requirement for TBK1 in responses to different vaccine platforms (mRNA, protein, viral vector)

This research direction could yield valuable insights for optimizing vaccine design, particularly for populations with suboptimal immune responses.