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HTRF Human and Mouse Total TBK1 Detection Kit, 500 Assay Points

The total TBK1 kit detects cellular TBK1 and can be combined with our phospho-TBK1 kit for an optimal readout of the TLR3/4, cGAS-STING pathways

For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption & disposal requirements under European REACH regulations (EC 1907/2006).

Product Variants
Part number: 64NTBPEG
Unit Size: 500 Assay Points
List price: USD 2,147.00
Your price:
USD 2,147.00
USD 2,147.00 /each
Part number: 64NTBPEH
Unit Size: 10,000 Assay Points
List price: USD 12,490.00
Your price:
USD 0.00
USD 12,490.00 /each

Overview

The Total TBK1 cellular assay is designed to monitor the expression level of TBK1 proteins, both phosphorylated and unphosphorylated. It is compatible with our phospho-TBK1 kit and enables the analysis of phosphorylated and total proteins from a single sample for a better readout of T-cell activation. 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.

Specifications

Assay Points
500
Assay Target Type
Kit
Assay Technology
HTRF
Brand
HTRF
Quantity
1
Therapeutic Area
Oncology & Inflammation
Unit Size
500 Assay Points

Video gallery

How it works

Total TBK1 assay principle

The total TBK1 assay quantifies the expression level of TBK1 in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The total TBK1 assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of TBK1 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the proteins expression under a no-wash assay format.

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Total TBK1 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 Total TBK1 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

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Total TBK1 1-plate assay protocol

Detection of total TBK1 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.

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Assay validation

Analysis of total TBK1 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.

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Analysis of total TBK1 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 23-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 23-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.

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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.

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Simplified pathway

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 23-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.

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SDS, COAs, Manuals and more

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