Revvity, 64ATG16S8PEG, HTRF Human and Mouse Phospho-ATG16L1 (Ser278) Detection Kit, 500 Assay Points

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 the 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.
HTRF assays offer many advantages over other technologies:

Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
How it works
HTRF Phospho-ATG16L1 (Ser278) assay principle
The HTRF Phospho-ATG16L1 (Ser278) assay measures ATG16L1 when phosphorylated at Serine 278. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The HTRF Phospho-ATG16L1 (Ser278) assay uses two 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 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 protein’s phosphorylation state under a no-wash assay format.

 

HTRF Phospho-ATG16L1 (Ser278) two-plate assay protocol
The two-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 HTRF Phospho-ATG16L1 (Ser 278) detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

HTRF Phospho-ATG16L1 (Ser278) one-plate assay protocol
Detection of phospho ATG16L1 (Ser 278) 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. Application-Cell Signaling
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 1 Lysis Buffer 2 Lysis Buffer 3 Lysis Buffer 4
Molecular Modification-Phosphorylation
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Phosphoproteins
Target Species-Human Mouse
Technology-TR-FRET
Unit Size-500 assay points