Phospho-TBK1 (Ser172) & Total TBK1 Assay Kits

Phospho-TBK1 (Ser172) and TBK1 assays to monitor the activation of TLR3/4 and cGAS-STING signaling pathways

These cell-based assays are designed to monitor the expression of TBK1, as well as its phosphorylation on Ser172 which is a hallmark of its activation.

Following pathogen infection and binding of dsRNA, LPS, or dsDNA to their respective cell surface receptors TLR3, TLR4 or cytoplasmic sensor cGAS, the kinase TBK1 is recruited to signaling complexes and activated by autophosphorylation at Ser172. The kinase in turn activates IRF3, which translocates to the nucleus and induces the transcription of type I IFNs, leading to an inflammatory response.

TBK1 is also involved in autophagy, where it directly phosphorylates the autophagy receptors optineurin and p62, which target the protein cargo to the autophagosome.

In NASH (Non-Alcoholic Steatohepatitis)/liver fibrosis, Kupffer cells are continuously exposed to an overload of gut-derived bacterial LPS, leading to the upregulation of TLR4, and therefore to an overactivation of TBK1. Therapeutic strategies consist in inhibiting this pathway.

The cGAS-STING signaling axis has recently emerged as a key player in immuno-oncology. Therapeutic activation of this pathway, which can be monitored by an increase in TBK1 phosphorylation, has shown promising anti-tumor effects in vivo.

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Assay principle

HTRF® - the homogeneous cell-based sandwich immunoassay

Cisbio Bioassay’s Phospho- and Total TBK1 assays are based on a TR-FRET sandwich immunoassay format comprising two antibodies, one labeled with Eu3+cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind to phospho-TBK1 or total TBK1, and the proximity of donor and acceptor then leads to a fluorescent TR-FRET signal. The protocol is optimized for a 96- or 384-well low volume microplate format (20 µL final), but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

The assay can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho- and total TBK1 can be quantitatively detected using the HTRF Phospho-TBK1 (S172) and Total TBK1 cellular assay kit reagents and most TR-FRET multimode plate readers.

A simpler, more flexible assay protocol - adapted to your applications

Two-plate assay protocol

For added flexibility, the assays can be run under a two-plate assay protocol, where cells are plated, treated, and lysed in a culture plate. For detection, lysates are subsequently transferred into a 96- or 384-well low volume detection microplate where the HTRF® reagents are added. This also enables monitoring of the cells' viability and confluence in an appropriate cell culture plate.

What to expect at the bench

This video covers the principles of phospho-HTRF assays and gives guidelines for performing them, using our phospho-ERK assay as an example.

Guidelines and tips to get the best out of your assays

Guidelines for cell culture and Lysis to prior detection, Download tehcnical note

Download our cell-based phospho-protein data normalization application note

Product performances

1. 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 next day, the 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 the addition of 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, demonstrating that LPS does not induce any cytotoxic effect.

2. 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 next day, the 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 the addition of 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.

3. 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 the HTRF® Phospho- or Total TBK1 kits, 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 twice as sensitive as the Western Blot technique.

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 the 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. Similarly, 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.

Following 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.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
TBK1 total kit - 500 tests64NTBPEG
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TBK1 total kit - 10,000 tests64NTBPEH
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TBK1 total kit - 50,000 tests64NTBPEY
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TBK1 phospho-S172 kit - 500 tests64TBKPEG
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TBK1 phospho-S172 kit - 10,000 tests64TBKPEH
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TBK1 phospho-S172 kit - 50,000 tests64TBKPEY
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Companion products

DescriptionCat. noProduct insertMSDS
TBK1 total kit - control lysate64NTBTDA
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TBK1 phospho-S172 kit - control lysate64TBKTDA
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