Alpha-Tubulin Housekeeping Cellular Kit
Phospho-TBK1 (Ser172) cellular kit HTRF®
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
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High sensitivity
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Faster and more convenient than ELISA
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Low sample consumption
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
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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 proteins phosphorylation state under a no-wash assay format.
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) 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.
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 doesnt induce any cytotoxic effect.
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 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.
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.
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.
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
Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers
HTRF cellular phospho-protein assays
Physiologically relevant results fo fast flowing research - Flyers
Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers
PI3K/AKT/mTor translational control pathway - 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
From 2D, 3D cell cultures to xenografts: A smart HTRF platform to maximize anticancer drug discovery
One technology across all samples - Application Notes
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
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
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
A single technology for 2D cells, 3D cells, and xenograft models - 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
Cell-based kinase assays in HTS ? potential and limitations for primary and secondary screening
In collaboration with Bayer - Scientific Presentations
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
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
HTRF HMGB1, IL-1β and IL-18 assays discriminate between inflammasome triggered pyroptosis, and necroptosis - Application Notes
Advance your research on Fibrosis
Kits and reagents for Fibrosis research - Flyers
Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection
Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes
Assessment of drug efficacy and toxicity by combining innovative technologies
Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes
Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)
Learn how to reduce time and sample consumption - Application Notes
Tacking autophagy regulation research a step further - Guides
Safety Data Sheet (DEU) TBK1 P-S172 Kit / 64TBKPEG
64TBKPEG - Safety Data Sheet
Safety Data Sheet (ELL) TBK1 P-S172 Kit / 64TBKPEG
64TBKPEG - Safety Data Sheet
Safety Data Sheet (FRA-FR) TBK1 P-S172 Kit / 64TBKPEG
64TBKPEG - Safety Data Sheet
Safety Data Sheet (ITA) TBK1 P-S172 Kit / 64TBKPEG
64TBKPEG - Safety Data Sheet
Safety Data Sheet (SPA) TBK1 P-S172 Kit / 64TBKPEG
64TBKPEG - Safety Data Sheet
Safety Data Sheet (ENG-GB) TBK1 P-S172 Kit / 64TBKPEG
64TBKPEG - Safety Data Sheet
Safety Data Sheet (ENG-US) TBK1 P-S172 Kit / 64TBKPEG
64TBKPEG - Safety Data Sheet
Safety Data Sheet (DEU) TBK1 P-S172 Kit / 64TBKPEH
64TBKPEH - Safety Data Sheet
Safety Data Sheet (ELL) TBK1 P-S172 Kit / 64TBKPEH
64TBKPEH - Safety Data Sheet
Safety Data Sheet (FRA-FR) TBK1 P-S172 Kit / 64TBKPEH
64TBKPEH - Safety Data Sheet
Safety Data Sheet (ITA) TBK1 P-S172 Kit / 64TBKPEH
64TBKPEH - Safety Data Sheet
Safety Data Sheet (SPA) TBK1 P-S172 Kit / 64TBKPEH
64TBKPEH - Safety Data Sheet
Safety Data Sheet (ENG-GB) TBK1 P-S172 Kit / 64TBKPEH
64TBKPEH - Safety Data Sheet
Safety Data Sheet (ENG-US) TBK1 P-S172 Kit / 64TBKPEH
64TBKPEH - Safety Data Sheet
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