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HTRF Human and Mouse Total ATG16L1 Detection Kit HTRF®

This HTRF kit enables the cell-based quantitative detection of total ATG16L1 as a readout of the autophagy pathway, and can be combined with our Phospho-ATG16L1 (Ser278) kit.

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  • All inclusive kit All inclusive kit
  • Low sample consumption Low sample consumption
  • No-wash No-wash
  • High sensitivity High sensitivity

This HTRF kit enables the cell-based quantitative detection of total ATG16L1 as a readout of the autophagy pathway, and can be combined with our Phospho-ATG16L1 (Ser278) kit.

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Overview

Autophagy Related 16 Like 1 (ATG16L1) is a protein that is essential for autophagy. ATG16L1 is recruited to the phagophore and recruits ATG5-ATG12 complex to a phagophore, facilitating LC3 lipidation (LC3-I to LC3-II conversion) and contributing to autophagosome maturation.

Phosphorylated ATG16L1 at Ser278 is only present on newly-forming autophagosomes, making its quantification an ideal surrogate for autophagic vesicle formation and autophagy induction. ATG16L1 is involved in conditions such as Alzheimer’s Disease, colorectal cancer, heart disease, breast cancer, and chronic myeloid leukemia.

Benefits

  • SPECIFICITY
  • PRECISION
  • LOW SAMPLE CONSUMPTION

HTRF Total ATG16L1 assay principle

The HTRF Total ATG16L1 assay quantifies the expression level of ATG16L1 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based, and does not require gels, electrophoresis, or transfer.

The HTRF Total ATG16L1 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 ATG16L1 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 protein’s expression under a no-wash assay format.

Principle of the HTRF Total ATG16L1 assay

HTRF Total ATG16L1 two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a low volume detection plate (either HTRF 384-lv or 96-lv plate) before the addition of HTRF Total ATG16L1 detection reagents. This protocol enables the cells' viability and confluence to be monitored

Two-plate protocol of the HTRF Total ATG16L1 assay

HTRF Total ATG16L1 one-plate assay protocol

Detection of Total ATG16L1 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 Total ATG16L1 assay

Validation of HTRF Total ATG16L1 and Phospho-ATG16L1 (Ser278) kits on the human U-87 MG cell line using mTOR and ULK1/2 inhibitors

Human U-87 MG  cells  were plated in a 96-well plate (100,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with a dose-response of AZD2014, an mTOR inhibitor, for 3 h at 37°C, 5% CO2. Following culture medium removal, cells were lysed with 25 µl of supplemented lysis buffer #3 (1X) + BR (2X) for 1 h at RT under gentle shaking. After cell lysis, 16 µL of lysate were transferred into a 384-well low volume white microplate, then 4 µL of the HTRF Phospho-ATG16L1 (Ser 278) or Total ATG16L1 detection reagents were added. The HTRF signals for the two kits were recorded after an overnight incubation at room temperature.


As expected, AZD2014, a potent mTOR inhibitor, induced autophagy activation, leading to a dose-dependent increase in ATG16L1 phosphorylation on Serine 278 without any significant effect on the expression level of the total ATG16L1 protein.

Validation of HTRF Total ATG16L1 and Phospho-ATG16L1 (Ser278) kits on the human U-87 MG cell line using mTOR and ULK1/2 inhibitors

Human U-87 MG  cells  were plated in a 96-well plate (100,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with 2.5 µM of AZD2014, an mTOR inhibitor, and  a dose-response of ULK-101, an autophagy kinase ULK1/2 inhibitor, for 3 h at 37°C, 5% CO2. Following culture medium removal, cells were lysed with 25 µl of supplemented lysis buffer #3 (1X) + BR (2X) for 1 h at RT under gentle shaking. After cell lysis, 16 µL of lysate were transferred into a 384-well low volume white microplate, then 4 µL of the HTRF Phospho-ATG16L1 (Ser 278) or Total ATG16L1 detection reagents were added. The HTRF signals for the two kits were recorded after an overnight incubation at room temperature.


As expected, ULK-101, a potent and dual autophagy kinase ULK1/2 inhibitor, repressed autophagy activation induced by AZD2014, leading to a dose-dependent decrease in ATG16L1 phosphorylation on Serine 278, without any significant effect on the expression level of the ATG16L1 total protein.

Validation of HTRF Total ATG16L1 and Phospho-ATG16L1 (Ser278) kits on the human U-87 MG cell line using mTOR and ULK1/2 inhibitors

Specificity of HTRF Total ATG16 assay using HAP1 ATG16 knock-out cells

The human total ATG16L1 expression level was assessed using the HTRF Total kit in Wild Type (WT) and ATG16 knock-out (KO) HAP1 cells. 


The cell lines were cultured in flasks and incubated 48 h at 37°C, 5% CO2. After culture medium removal, cells were lysed with supplemented lysis buffer #3 (1X) + BR (2X) for 30 min at RT under gentle shaking.  After cell lysis, 16 µL of lysate were transferred into a 384-well low volume white microplate then 4 µL of the HTRF Total ATG16L1 detection reagents were added. In parallel, GAPDH level was monitor using the GAPDH Housekeeping Cellular Kit (# 64GAPDHPET/G/H). 4 µL of cell lysate were prediluted with 180 µL of diluent #11. Then, 16 µL of this diluted material were mixed with 4 µL of the HTRF GAPDH Housekeeping Cellular detection reagents. HTRF signal for both kits was recorded after an overnight incubation at room temperature.


The measured level of GAPDH was equivalent under both tested conditions. In contrast, while the HTRF Total ATG16L1 signal was significantly positive in HAP1 WT cells, it was dramatically decreased (equivalent to the negative signal) in HAP1 ATG16 KO cells, demonstrating the specificity of the HTRF Total ATG16L1 assay for the detection of the ATG16L1 protein.


HAP1 ATG16 KO (10bp deletion) cell line from Horizon Discovery # HZGHC000296c010

HAP1 WT cell line from Horizon Discovery # C631

Specificity of HTRF Total ATG16 assay using HAP1 ATG16 knock-out cells

Versatility of HTRF Total ATG16L1 assay in cell lines from multiple species

Human (PC-3), mouse (C2C12) and rat (H4-II-E) cell lines were seeded at 100,000 cells/well in a 96-well plate. After a 24h incubation, the cells were lysed with 25 µL of supplemented lysis buffer #3 (1X) + BR (2X) for 1 h at RT under gentle shaking. After cell lysis, 16 µL of lysates were transferred into a 384-well low volume white microplate then 4 µL of the HTRF Total ATG16L1 detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.


The HTRF Total ATG16L1 assay efficiently displayed significant positive signals in the cell lines from the 3 species tested, demonstrating its capability to detect human, mouse and rat versions of the protein, and its suitability for translational research.

Versatility of HTRF Total ATG16L1 assay in cell lines on multiple species

HTRF Total ATG16L1 Detection Kit compared to Western Blot

HCT116 cells were cultured in flasks and incubated 48 h at 37°C, 5% CO2. Then, the cells were treated with 100 nM of Calyculin A for 10 min. After culture medium removal, cells were lysed with supplemented lysis buffer #3 (1X) + BR (2X) for 30 min at RT under gentle shaking.  Soluble supernatants were collected after 10 min centrifuging. Equal amounts of lysates were used for a side-by-side comparison of WB and HTRF.


The HTRF Total ATG16L1 assay is 4 times more sensitive than Western Blot assay.

Comparison between HTRF and WB sensitivity on phospho-ATG16L1 (Ser278)

Simplified ATG16 signaling pathway in macroautophagy

Autophagy is an evolutionary conserved cellular process that occurs in virtually all eukaryotic cells, ranging from yeast to mammals. Autophagy is crucial in the maintenance of homeostasis, being involved in numerous physiological processes including stress responses (e.g. starvation, hypoxia, high temperature), cell growth, and aging. Conversely, dysfunctions in autophagic mechanisms have been associated with diseases such as cancer, neurodegenerative diseases, infectious diseases, and cardiac and metabolic diseases.

There are 3 types of autophagy: macroautophagy, microautophagy and chaperone-mediated autophagy.


Macroautophagy (hereafter referred to as autophagy) starts with the formation of a bud from lipid phosphatidylinositol 3-phosphate rich membranes such as reticulum endoplasmic, Golgi, or plasma membranes, and known as a pre-autophagosomal structure (PAS). This cup-shaped structure requires the ULK1/Atg1 complex formed by Atg13, Atg101, FIP200, and ULK1 proteins. A transmembrane protein, Atg9, contained in vesicles is involved in the recruitment of Atg2-Atg18 to the PAS. With its phospholipid transfer activity, the Atg2 protein supplies phospholipids necessary for membrane elongation. ULK1 phosphorylates and activates Beclin1, which partners with Vsp15, Vsp34, Atg14, and NRFB2/Atg38 to form a class III Phosphatidylinositol 3-kinase (PI3KC3-Complex 1). This complex leads to the production of phospho-inositol triphosphate (PIP3), which further directs the recruitment of PI3P binding Atg18/WIPI 1–4 proteins and the Atg12-Atg5-Atg16 complex. The latter complex, along with Atg4 and Atg7, is implicated in the LC3B lipidation (LC3-I to LC3-II conversion) on the phagophore. This contributes to autophagosome maturation, which subsequently fuses with lysosomes forming an autolysosome, where trapped cellular components are degraded and building blocks recycled.


Phosphorylated ATG16L1 at Ser278 is only present on newly-forming autophagosomes, making its quantification an ideal surrogate for autophagic vesicle formation and autophagy induction. Importantly, its levels are not affected by prolonged stress or late-stage autophagy blocks, which can jeopardize autophagy analysis. Moreover, ATG16L1 phosphorylation by the autophagy kinase ULK1 is a conserved indicator of autophagy induction, which is activated by multiple stimuli.

Taken together, analysis of ATG16L1 phosphorylation represents an excellent readout when studying autophagy induction.

Simplified ATG16 signaling pathway in macroautophagy

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 - Technical Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

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

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

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

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

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

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

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

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

Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Application 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

Manual ATG16L1 Total Kit / 64ATG16TPEG-64ATG16TPEH

64ATG16TPEG-64ATG16TPEH - Product Insert

Certificate of Analysis ATG16L1 Total Kit / 64ATG16TPEG

64ATG16TPEG Batch 01A - Quality Control Report

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