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HTRF Human and Mouse Phospho-ATG14 Ser29 Detection Kit

HTRF Human and Mouse Phospho-ATG14 Ser29 Detection Kit HTRF®

This HTRF kit enables the cell-based quantitative detection of ATG14 phosphorylation at Ser29 as a readout of the autophagy pathway.

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All inclusive kit
Low sample consumption
No-wash
High sensitivity

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This HTRF kit enables the cell-based quantitative detection of ATG14 phosphorylation at Ser29 as a readout of the autophagy pathway.

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Overview

ATG14, Autophagy related protein 14, or BAKOR for Beclin 1-associated autophagy-related key regulator, is a key player in the autophagosome nucleation step in macroautophagy. Upon cellular stress, the nutrient/energy-sensitive sensors mTOR and AMPK lead to the activation of the ULK1 complex, which allows phosphorylation of ATG14 on Serine 29, in turn enabling the downstream activation of the PIK3C3 autophagosome nucleation complex.

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HTRF Phospho ATG14 assay principle

The HTRF Phospho-ATG14 (Ser29) assay measures ATG14 when phosphorylated at Ser 29. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer.

The Phospho-ATG14 (Ser29) assay uses 2 labeled antibodies: one with a donor fluorophore, the other with an acceptor. The first antibody was selected for its specific binding to the phosphorylated motif on the protein, and the second for its ability to recognize the protein independently of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving the two labeled antibodies 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.

Principle of the HTRF phospho-ATG14 (Ser29) assay

HTRF Phospho-ATG14 Ser29 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of the Phospho-ATG14 Ser 29 HTRF detection reagents.

This protocol enables the cells' viability and confluence to be monitored.

Two-plate protocol of the HTRF phospho ATG14 assay

HTRF Phospho-ATG14 Ser29 one-plate assay protocol

Detection of phospho ATG14 Ser 29 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.

One-plate protocol of the HTRF phospho ATG14 assay

ATG14 signaling pathway in macro-autophagy

Cellular Autophagy is a specialized degradation and recycling process that is instrumental for cell homeostasis, being activated in response to several different stresses. There are 3 types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy. Macroautophagy pathways involve several key steps: initiation, nucleation, elongation of phagophore complexes, then sequestration of cytoplasmic cargos with LC3-PE recruitment followed by fusion with lysosome yielding to cargo degradations.

Biogenesis of the autophagosome is controlled by sequential and concerted actions of the so-called autophagy related proteins, ATGs, which are activated and recruited to the ER and autophagosome membranes. The recruitment of the ULK1 complex is the first event in the initiation step. ULK1 is a Ser/Thr kinase which forms a complex with the Atg13, Atg101 and FIP200 proteins. This complex is the most upstream component of the core autophagy machinery and is therefore the key initiator of autophagy in mammalian cells. ULK1 is regulated by the key nutrient/energy-sensitive kinases mTOR and AMPK, which are both able to phosphorylate ULK1 on Serine 317 and Serine 556, and directly regulate its kinase activity.

The Activated ULK1 complex then binds ATG14 via ATG13, and phosphorylates ATG14 on Serine 29. The kinase activity of phosphorylated and activated ATG14 stimulates other proteins of the PIK3C3 complex (nucleation) that is responsible for the critical step of phosphorylation of phosphatidylinositols (PI) into phosphatidylinositol-3-phosphate (PI3P). This in turn is responsible for the formation of the initial phagosomal membrane structure and later allows fixation of LC3-II using other ATG proteins (ATG16/12/5 complex), thus generating a support for elongation and closing steps.

This means tha phosphorylation of ATG14 protein on serine 29 is a key early marker of the nucleation step in the engagement of the macroautophagic process.

ATG14 signaling pathway in macro-autophagy

HTRF cellular phospho-protein assays

physiologically relevant results fo fast flowing research - Flyers

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

Insider Tips for successful sample treatment - Notes techniques

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Notes techniques

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Notes techniques

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF® phospho-protein platform facilitates the dissection of signaling pathways involved in insulin resistance and metabolic disorders

Multi-tissue cellular modeling and anlysis of insulin signaling - Posters

HTRF® cell signaling platform combined with iCell® Hepatocytes

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

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Implement HTRF phospho-protein assays at every step of the drug discovery process, from in vitro to in vivo models

A single technology for 2D cells, 3D cells, and xenograft models - Posters

Simplify complex pathway dissection by combining the power of HTRF® cellular phospho-assays and the flexibility of the CyBi®-FeliX liquid handling system

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 - Notes d'application

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

Tumor xenograft analysis: HTRF versus Western blot - Notes d'application

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Notes d'application

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

Increased flexibility of phospho-assays - Notes d'application

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Notes d'application

Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Notes d'application

How to run a cell based phospho HTRF assay

What to expect at the bench - Vidéos

Cell-based kinase assays in HTS ? potential and limitations for primary and secondary screening

In collaboration with Bayer - Présentations scientifiaues

Unleash the potential of your phosphorylation research with HTRF

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

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Vidéos

Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection

Seeding and lysing recommendations for a number of cell culture vessels. - Notes techniques

Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Notes d'application

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 - Notes d'application

Autophagy Regulation eBook

Tacking autophagy regulation research a step further - Guides

Manual ATG14 p-S29 Kit / 64ATG14S9PEG-64ATG14S9PEH

64ATG14S9PEG-64ATG14S9PEH - Protocoles

Certificate of Analysis ATG14 p-S29 Kit / 64ATG14S9PEG

64ATG14S9PEG Lot 01B - Rapports de contrôle qualité

Safety Data Sheet (DEU) ATG14 p-S29 Kit / 64ATG14S9PEG

64ATG14S9PEG - Fiches de sécurité

Safety Data Sheet (ELL) ATG14 p-S29 Kit / 64ATG14S9PEG

64ATG14S9PEG - Fiches de sécurité

Safety Data Sheet (FRA-FR) ATG14 p-S29 Kit / 64ATG14S9PEG

64ATG14S9PEG - Fiches de sécurité

Safety Data Sheet (ITA) ATG14 p-S29 Kit / 64ATG14S9PEG

64ATG14S9PEG - Fiches de sécurité

Safety Data Sheet (SPA) ATG14 p-S29 Kit / 64ATG14S9PEG

64ATG14S9PEG - Fiches de sécurité

Safety Data Sheet (ENG-GB) ATG14 p-S29 Kit / 64ATG14S9PEG

64ATG14S9PEG - Fiches de sécurité

Safety Data Sheet (ENG-US) ATG14 p-S29 Kit / 64ATG14S9PEG

64ATG14S9PEG - Fiches de sécurité

Safety Data Sheet (DEU) ATG14 p-S29 Kit / 64ATG14S9PEH