TBK1 Antibody, HRP conjugated

What is TBK1 and why is it an important research target?

TBK1 is a serine-threonine protein kinase that functions as a signaling hub in multiple cellular pathways. It plays crucial roles in antiviral innate immunity, cell survival, and proliferation in both tumor microenvironments and tumor cells . TBK1 is also implicated in the regulation of autophagy through phosphorylation of several autophagy proteins, including Optineurin and the C9ORF72/SMCR8 complex . Mutations in TBK1 have been linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), making it an important research target for neurological disease studies . Recent research has also identified TBK1 as a potential therapeutic target in cancer, particularly in head and neck cancer (HNC) treatment .

What is the molecular structure and weight of human TBK1?

Human TBK1 has a calculated molecular weight of approximately 84 kDa based on its amino acid sequence . The protein is encoded by the TBK1 gene (Uniprot accession # Q9UHD2) . When detected by Western blot techniques such as Simple Western, TBK1 appears as a specific band at approximately 90 kDa . The protein consists of several functional domains, including a kinase domain, ubiquitin-like domain, and coiled-coil domains that mediate protein-protein interactions and regulate its activity.

What is the principle behind HRP conjugation to TBK1 antibodies?

HRP (Horseradish peroxidase) conjugation to antibodies involves chemical linking of the enzyme to the antibody while maintaining both antibody specificity and enzyme activity. The conjugation creates a detection tool where the antibody provides specificity for TBK1, and the HRP enzyme generates a detectable signal through its catalytic activity . The most effective conjugation methods preserve the antigen-binding capacity of the antibody while ensuring high enzymatic activity of HRP. Several approaches exist for this conjugation, including reductive amination, maleimide-thiol coupling (using SMCC and 2-MEA), and more advanced methods like SoluLINK bioconjugation technology that uses pre-activated HRP with 4-formylbenzamide (4FB) .

What are the validated applications for TBK1 antibodies and what dilutions are recommended?

TBK1 antibodies have been validated for multiple applications including Western blot (WB), immunoprecipitation (IP), immunofluorescence/immunocytochemistry (IF/ICC), and enzyme-linked immunosorbent assay (ELISA) . The recommended dilutions vary by application:

For HRP-conjugated TBK1 antibodies specifically, the dilution may need optimization based on the conjugation efficiency and the specific detection system used .

How should I validate a TBK1 antibody for specificity in my experimental system?

Validation of TBK1 antibodies should include positive and negative controls to confirm specificity. An optimal approach involves using:

• Wild-type (WT) cells/tissues expressing TBK1

• Isogenic knockout (KO) cells lacking TBK1 expression

• Overexpression systems with tagged TBK1

Researchers have successfully validated TBK1 antibodies in U2OS cells (with CRISPR/Cas9-mediated TBK1 knockout as negative control) across multiple applications . For immunofluorescence validation, a mixed-culture approach where WT cells (labeled with one fluorescent dye) and TBK1 KO cells (labeled with a different dye) are co-cultured provides an excellent control system within the same field of view . For Western blot validation, running parallel samples from WT and KO cells with Ponceau staining ensures equal loading and transfer efficiency .

What cell types and tissues show significant TBK1 expression for antibody validation?

TBK1 shows differential expression across tissues and cell types. Notable expression has been documented in:

• Human prostate cancer tissue (cytoplasm of epithelial cells)

• Human islets, predominantly in β-cells

• Daudi human Burkitt's lymphoma cells

• HeLa human cervical epithelial carcinoma cells

• U2OS human osteosarcoma cells

• Human head and neck cancer cells

For antibody validation, U2OS cells have been particularly useful as their expression of TBK1 represents an average range found in cancer cells, and they are amenable to CRISPR/Cas9 modification for generating knockout controls .

What are the optimal methods for preparing high-quality TBK1 antibody-HRP conjugates?

Preparing high-quality TBK1 antibody-HRP conjugates requires careful consideration of conjugation chemistry and purification methods. The SoluLINK bioconjugation technology offers significant advantages by using pre-activated HRP with stable 4-formylbenzamide (4FB) groups that react specifically with hydrazine-modified antibodies . This approach maintains high enzyme activity and preserves antibody specificity. The key steps include:

• Antibody modification with HyNic (6-hydrazinonicotinamide)

• HRP activation with 4FB

• Conjugation reaction catalyzed by TurboLINK catalyst buffer

• Purification using specialized spin columns to remove unconjugated HRP

The method is performed under gentle pH conditions (6.0-7.4) without harsh chemicals or reducing agents, which helps maintain the structural integrity and functionality of both the antibody and enzyme . The resulting conjugates show excellent performance in ELISA and other applications, with high signal-to-noise ratios.

How should I analyze TBK1 phosphorylation status using HRP-conjugated antibodies?

Analyzing TBK1 phosphorylation (particularly at Ser172, which indicates activation) requires careful experimental design:

• Stimulate cells with appropriate activators (e.g., poly(I:C) for viral RNA mimicry, which activates TBK1 in a time-dependent manner)

• Prepare cell lysates using phosphatase inhibitor-containing buffers to preserve phosphorylation status

• Perform Western blot analysis using:

  • A phospho-specific TBK1 antibody (detecting pSer172)

  • A total TBK1 antibody to normalize expression levels

  • Appropriate HRP-conjugated secondary antibodies if using unconjugated primaries

For quantification, calculate the ratio of phosphorylated to total TBK1. When using directly HRP-conjugated TBK1 antibodies, ensure the conjugation process hasn't affected the phospho-epitope recognition. Control experiments should include phosphatase treatment of some samples and time-course activation studies .

What are the most effective blocking and washing conditions when using TBK1 antibody-HRP conjugates?

Optimizing blocking and washing conditions is critical for maximizing signal-to-noise ratio when using TBK1 antibody-HRP conjugates:

For phospho-TBK1 detection, BSA is generally preferred over milk as blocking agent because milk contains phospho-proteins that may increase background . Additionally, including 1 mM sodium orthovanadate in wash buffers helps preserve phosphorylation during the washing steps.

How can TBK1 antibodies be used to investigate the role of TBK1 in selective autophagy?

TBK1 plays a crucial role in selective autophagy through phosphorylation of autophagy receptors such as Optineurin (OPTN), p62/SQSTM1, and the C9ORF72/SMCR8 complex . To investigate these interactions:

• Co-immunoprecipitation studies: Use TBK1 antibodies to pull down TBK1 and analyze co-precipitated autophagy proteins. This approach successfully identified p62/SQSTM1, TAX1BP1, OPTN, and other autophagy-related proteins as TBK1 interactors .

• Phosphorylation analysis: TBK1 phosphorylates the LIR (LC3-interacting region) motif of OPTN, enhancing its interaction with LC3B and promoting autophagosome formation . Use phospho-specific antibodies against these targets alongside TBK1 detection.

• Colocalization studies: Combine TBK1 antibodies with markers for autophagosomes (LC3), autophagy receptors (p62, OPTN), and ubiquitinated proteins to visualize recruitment to autophagy substrates.

• Functional assays: Compare autophagic flux in wild-type versus TBK1-deficient cells using autophagy substrate clearance assays, particularly following specific stimuli like poly(I:C) treatment .

Recent research has shown that TBK1 facilitates autophagosome-lysosome fusion and selective clearance of ubiquitinated proteins, particularly under stress conditions, making this a rich area for investigation .

What experimental approaches can determine the role of TBK1 in cellular stress responses using TBK1 antibodies?

TBK1 has recently been identified as a key player in cellular stress responses, particularly in stress granule (SG) formation and stress-adaptive mechanisms . To investigate these functions:

• Stress induction and TBK1 activation: Apply various stressors (proteotoxic stress, HSP90 inhibition, or ubiquitin stress) and monitor TBK1 activation using phospho-specific antibodies (pSer172).

• Stress granule analysis: Combine TBK1 immunofluorescence with stress granule markers (G3BP1) to analyze colocalization and recruitment kinetics during stress response.

• Pharmacological inhibition: Compare stress responses in the presence and absence of TBK1 inhibitors like GSK8612, which has shown significant inhibition of head and neck cancer tumorigenesis in xenografts .

• Quantitative proteomics: Immunoprecipitate TBK1 under various stress conditions and perform mass spectrometry to identify stress-specific interaction partners.

Recent research has revealed that TBK1 is required for stress granule formation and cellular protection under stress conditions. Additionally, MAP1LC3B (an autophagy marker) has been found partially localized within stress granules, suggesting a link between autophagy machinery and stress granule formation mediated by TBK1 .

How can TBK1 antibodies be used to investigate the role of TBK1 in immune cell function and disease models?

TBK1 plays crucial roles in both innate immunity and adaptive immune responses. Recent research has uncovered unexpected functions of TBK1 in B cell immunity . To investigate TBK1 in immune contexts:

• B cell germinal center (GC) formation: Use TBK1 antibodies to track expression and phosphorylation status during B cell differentiation stages. B cell-intrinsic TBK1 has been identified as crucial for GC formation, where it negatively regulates CD40 and BCR signaling to control IRF4 and c-Myc expression in Pre-GC B cells .

• Signaling pathway analysis: Monitor TBK1's impact on noncanonical NF-κB, TRAF2, and AKT phosphorylation in immune cells following receptor engagement.

• Disease models: In models of inflammatory diseases or viral infections, track TBK1 activation status in various immune cell populations using flow cytometry with intracellular phospho-TBK1 staining.

• Human TBK1 deficiency studies: Four patients with complete TBK1 deficiency have been identified who suffer from chronic and systemic autoinflammation driven by TNF-induced regulated cell death (RCD). Anti-TNF treatment improved their clinical condition, highlighting a role for TBK1 in suppressing inflammatory TNF-mediated cell death .

• Drug testing: Evaluate the effects of TBK1 inhibitors like WEHI-112 on immune responses, particularly in the context of antibody-dependent arthritis and other inflammatory conditions .

What are common issues when working with TBK1 antibody-HRP conjugates and how can they be resolved?

Common issues with TBK1 antibody-HRP conjugates include:

For TBK1 detection specifically, some antibodies may require optimization beyond manufacturer recommendations. For example, antibodies 28397-1-AP, PA5-17478, 703154, ab40676, and ab109735 have been reported to require dilution to 1/10000 because the signal was too strong following the supplier's recommendations .

How can I determine the optimal fixation conditions for immunofluorescence detection of TBK1?

Optimal fixation conditions for TBK1 immunofluorescence detection depend on the subcellular localization and epitope accessibility:

• Paraformaldehyde fixation: 4% PFA for 10-15 minutes at room temperature works well for many TBK1 antibodies and preserves cytoplasmic localization .

• Methanol fixation: Ice-cold methanol for 10 minutes can provide better access to some epitopes and reduce cytoplasmic background, but may affect certain protein-protein interactions.

• Mixed fixation approach: For comprehensive detection, compare PFA fixation followed by permeabilization with 0.1-0.5% Triton X-100 versus methanol fixation.

To determine optimal conditions, prepare U2OS wild-type and TBK1 knockout cells with different fluorescent labels (e.g., green for WT, far-red for KO) and plate them together. This approach allows direct comparison of antibody specificity under various fixation conditions . TBK1 typically shows cytoplasmic localization, with specific staining in the cytoplasm of epithelial cells as documented in prostate cancer tissue .

What controls should be included when using TBK1 antibody-HRP conjugates in research studies?

Comprehensive controls for TBK1 antibody-HRP conjugate experiments should include:

• Specificity controls:

  • TBK1 knockout or knockdown samples

  • Competition with immunizing peptide when available

  • Secondary antibody-only controls (for indirect detection methods)

• Technical controls:

  • Loading controls (housekeeping proteins or total protein staining)

  • Positive control samples with known TBK1 expression (e.g., Daudi or HeLa cell lysates)

  • Ponceau staining of membranes to verify equal loading and transfer

• Biological controls:

  • Unstimulated vs. stimulated samples (e.g., poly(I:C) treatment to activate TBK1)

  • Time-course experiments to track phosphorylation dynamics

  • Wild-type vs. mutant TBK1 expression constructs

• HRP activity controls:

  • Direct substrate test of the conjugate to confirm enzymatic activity

  • Comparison with unconjugated primary + HRP-secondary antibody detection

For immunofluorescence applications, the cell-mixing approach described earlier (differentially labeled WT and KO cells) provides an excellent internal control system .

How might TBK1 antibodies contribute to understanding the role of TBK1 in neurodegenerative diseases?

TBK1 has been linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) through genetic studies identifying mutations in TBK1 . TBK1 antibodies can contribute to understanding these connections through:

• Mutation impact analysis: Compare wild-type and mutant TBK1 expression, localization, and phosphorylation status in patient-derived samples or model systems.

• Protein interaction studies: Investigate how TBK1 mutations affect interactions with key autophagy proteins (Optineurin, p62/SQSTM1, C9ORF72/SMCR8) that are also implicated in ALS/FTD .

• Cellular phenotype assessment: Analyze autophagy flux, mitochondrial clearance, and aggregate formation in neuronal models with TBK1 mutations.

• Biomarker development: Evaluate whether TBK1 phosphorylation status or protein levels in accessible biosamples correlate with disease progression.

• Therapeutic response monitoring: For experimental treatments targeting pathways involving TBK1, quantify changes in TBK1 activation or localization.

Recent studies have shown that TBK1 controls TNF-mediated inflammation, with TBK1-deficient patients developing autoinflammation driven by TNF-induced regulated cell death . This suggests examining neuroinflammatory aspects of neurodegeneration through TBK1-related mechanisms.

What are the latest methodological advances in TBK1 research that exploit antibody-based approaches?

Recent methodological advances in TBK1 research using antibody-based approaches include:

• Proximity labeling proteomics: BioID or APEX2 fusions with TBK1 coupled with antibody-based enrichment to identify dynamic, context-specific TBK1 interactomes.

• Live-cell imaging: Nanobody-based detection systems for visualizing TBK1 dynamics without fixation artifacts.

• Multi-omics integration: Combining phospho-TBK1 antibody-based proteomics with transcriptomics to map TBK1-dependent signaling networks.

• Single-cell analyses: Adaptation of phospho-TBK1 antibodies for CyTOF or single-cell western blotting to reveal heterogeneity in cellular responses.

• Quantitative spatial proteomics: Using TBK1 antibodies for multiplexed immunofluorescence or imaging mass cytometry to map spatial relationships between TBK1 and its substrates or regulators.

Research has revealed that TBK1 functions as a signaling hub coordinating stress-adaptive mechanisms, facilitating autophagosome-lysosome fusion, and regulating selective autophagic clearance . These advanced methodologies can help dissect these complex functions with greater precision.

How can TBK1 antibodies be used to investigate TBK1's role as a potential therapeutic target in cancer?

TBK1 has emerged as a promising therapeutic target, particularly in cancer contexts . TBK1 antibodies can contribute to target validation and drug development through:

• Expression profiling: Quantify TBK1 expression and activation across cancer types and correlate with clinical outcomes to identify cancer types likely to respond to TBK1 inhibition.

• Mechanism studies: Investigate how TBK1 promotes cancer cell survival through autophagy regulation and stress granule formation .

• Drug screening: Use phospho-TBK1 antibodies to monitor target engagement and pathway inhibition by candidate TBK1 inhibitors.

• Combination therapy assessment: Evaluate whether TBK1 inhibition sensitizes cancer cells to other treatments by measuring markers of cell death and stress responses.

• Biomarker development: Identify which TBK1-dependent pathways predict therapeutic response.