Revvity, 64ATMS1PEH, HTRF Human Phospho-ATM (Ser1981) Detection Kit, 10,000 Assay Points

Overview
This HTRF cell-based assay enables the rapid, quantitative detection of ATM phosphorylated at Serine 1981, as a readout of the ATM/CHK2 signaling pathway upon a DNA damage response (DDR). This kit is complementary to the phospho CHK2 Thr68 for studies of the ATM-CHK2 pathway.
In response to DNA damage, such as Double Strand Breaks (DSBs), the ATM€“Chk2 pathway and replication checkpoint responses are activated. These mediate G1/S checkpoints to arrest cell cycle progression and allow extra time for DNA repair.
In response to DNA DSBs, ATM autophosphorylates to generate the active monomeric kinase. Activation of ATM results in the phosphorylation of a diverse array of downstream targets such as H2A histone family member X, H2Ax, or the checkpoint kinase Chk2 and its downstream effectors.
How it works
HTRF phospho ATM assay principle
The Phospho-ATM (Ser1981) assay measures ATM when phosphorylated at Serine 1981. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer.
The Phospho-ATM (Ser 1981) 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 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-ATM Ser 1981 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 the addition of the phospho-ATM  ser 1981 HTRF detection reagents.
This protocol enables the cells' viability and confluence to be monitored.

 

HTRF Phospho-ATM Ser 1981 one-plate assay protocol
Detection of phospho ATM Ser 1981 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 4
Molecular Modification-Phosphorylation
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Phosphoproteins
Target Species-Human
Technology-TR-FRET
Unit Size-10,000 assay points