Revvity, 64ATG16TPEG, HTRF Human and Mouse Total ATG16L1 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 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.
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 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.

 

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

 

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. Application-Cell Signaling
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 1 Lysis Buffer 3 Lysis Buffer 4
Molecular Modification-Total
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