Phospho-TBK1 (Ser172) cellular kit

The phospho-TBK1 (Ser172) assay enables the cell-based quantitative detection of phosphorylated TBK1 on Ser172 as a readout of the TLR3/4, cGAS-STING pathways
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  • Ready-to-use Ready-to-use
  • High sensitivity High sensitivity
  • Faster and more convenient than ELISA Faster and more convenient than ELISA
  • Low sample consumption Low sample consumption
The phospho-TBK1 (Ser172) assay enables the cell-based quantitative detection of phosphorylated TBK1 on Ser172 as a readout of the TLR3/4, cGAS-STING pathways
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Overview

The phospho-TBK1 cell based assay kit conveniently and accurately quantifies phosphorylated TBK1 at Ser172. Following pathogen infection, TBK1 can be activated by bacterial LPS and pathogen nucleic acids which trigger the activation of TLR3/4 and cGAS-STING signaling axis. This phospho-TBK1 assay can be used from basic research through to preclinical drug discovery phases and contains everything you need. It offers increased throughput compared to ELISA/WB.

Benefits

  • SPECIFICITY
  • PRECISION

Phospho-TBK1 (Ser172) assay principle

The Phospho-TBK1 (Ser172) assay measures TBK1 when phosphorylated at Ser172. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-TBK1(Ser172) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein’s phosphorylation state under a no-wash assay format.
Phospho-TBK1 (Ser172) assay principle

Phospho-TBK1 (Ser172) 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding phospho-TBK1 (Ser172)HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-TBK1 (Ser172) 2-plate assay protocol

Phospho-TBK1 (Ser172) 1-plate assay protocol

Detection of phosphorylated TBK1 on serine 172 with HTRF reagents can be performed in a single plate used for culturing, stimulation and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.
Phospho-TBK1 (Ser172) 1-plate assay protocol

Analysis of TBK1 phosphorylation to monitor the LPS/TLR4 axis in Kupffer cells

Mouse Kupffer cells (ImKC cell line) were plated in 96-well plates (200,000 cells/well) in complete culture medium and incubated at 37°C - 5% CO2. The day after, cells were treated for 1 hour with increasing concentrations of LPS diluted in serum-free medium. After medium removal, cells were then lysed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred twice over into a low volume white microplate before adding 4 µL of the HTRF® phospho-TBK1 or total TBK1 detection antibodies. The HTRF signal was recorded after an overnight incubation at RT. LPS induces the activation of TLR4 signaling, leading to a 3-fold increase in TBK1 phosphorylation on Ser172. The expression level of the kinase remains stable which demonstrates that LPS doesn’t induce any cytotoxic effect.
TBK1 Phosphorylation on Mouse Kupffer cells (ImKC cell line) stimulated with LPS

Analysis of TBK1 phosphorylation to monitor the activation of the cGAS-STING pathway

Mouse Kupffer cells (ImKC cell line) were plated in 96-well plates (200,000 cells/well) in complete culture medium and incubated at 37°C - 5% CO2. The day after, cells were treated for 1 hour with increasing concentrations of 2’3’-cGAMP diluted in serum-free medium. After medium removal, cells were then lysed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred twice over into a low volume white microplate before adding 4 µL of the HTRF® phospho-TBK1 or total TBK1 detection antibodies. The HTRF signal was recorded after an overnight incubation at RT. Cell treatment with 2’3’-cGAMP leads to the activation of STING which in turn triggers a 3.6-fold increase in TBK1 phosphorylation on Ser172. As expected, the expression level of the kinase remains constant.
TBK1 Phosphorylation on Mouse Kupffer cells (ImKC cell line) stimulated with 2'3'-cGamp

HTRF phospho- & total TBK1 cellular assays compared to Western Blot

The human cervical cancer cell line HeLa was seeded in a T175 flask in complete culture medium, and incubated for 2 days at 37°C, 5% CO2 until 90% confluency was reached. Cells were then treated with 200 nM Calyculin A for 10 minutes and lysed with 3 mL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking. Soluble supernatants were collected after a 10-minute centrifugation. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF® phospho- or total TBK1 detection antibodies. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot. Using HTRF® Phospho- or Total TBK1 kit, only 1,250 cells/well were sufficient to detect a significant signal while 2,500 cells were needed for minimal ECL signal detection using Western Blot. The HTRF assays are therefore 2 times more sensitive than the Western Blot technique.
HTRF Phospho-TBK1 assay compared to WesternBlot
HTRF Total TBK1 quantification assay compared to WesternBlot

TLR3/4 and cGAS-STING simplified pathway

TBK1 (TANK-Binding Kinase 1, also known as NAK) is a multimeric kinase activated by autophosphorylation at Ser172. This kinase is a key signaling node between several pathways leading to inflammation and autophagy. Following pathogen infection, TBK1 can be activated by bacterial LPS and pathogen nucleic acids which trigger the activation of TLR3/4 and cGAS-STING signaling axis. Upon LPS binding to TLR4 at the cell surface, the receptor translocates to endosomes and recruits the protein TRAM which in turn activates the protein TRIF. In the same way, dsRNA entering the cell binds to endosomal TLR3 which directly activates TRIF. In the case of dsDNA, it enters the cell and binds to the cytoplasmic sensor cGAS, inducing the production of 2’3’-cGAMP which in turn activates the adaptor protein STING. Further to the activation of each of these pathways, TBK1 is recruited to different signaling complexes and activated by autophosphorylation at Ser172. The kinase in turn activates the transcription factor IRF3 which translocates to the nucleus and induces the expression of type I IFN genes, leading to inflammatory responses. TBK1 is also involved in autophagy where it directly phosphorylates the autophagy receptors optineurin and p62, which target the protein cargo to the autophagosome.
TLR3/4 and cGAS-STING signaling pathway
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Open R&D: Sanofi Access Platform

In collaboration with Sanofi - Scientific Presentations

Cisbio lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Save time and money

Switch to HTRF assays - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

Side-by-side comparison of HTRF, Western Blot, ELISA and AlphaScreen® SureFire®

Do all cell-based kinase assays perform similarly? - Posters

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Videos

STING HTRF offer to bridge innate and adaptive immunity

cGAS-STING signaling pathway from A to Z - Brochures

Product Insert TBK1 P-S172 Kit / 64TBKPEG-64TBKPEH

64TBKPEG-64TBKPEH - Product Insert

Cisbio Product Catalog 2019

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

Novel HTRF platform to delineate STING pathway

Explore the whole STING pathway with a single technology - Posters

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

Characterize STING agonists with HTRF human IFNb

Data about HTRF IFNb correlation with gene reporter assay and ELISA - Application Notes

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

An innate and adaptive immunity recap

Insight into the diversity of cells & signaling pathways - Guides

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