Revvity, 64AURBT2PEG, HTRF Human Phospho-Aurora B (Thr232) Detection Kit, 500 Assay Points

Overview
This HTRF cell-based assay conveniently and accurately detects human Aurora B protein (related to the AURKB gene) ubiquitously expressed in eukaryotes. Aurora B belongs to the serine/threonine Aurora kinase family (including Aurora A and Aurora C), essential for cell division via mitosis regulation especially in the process of chromosomal segregation. It is activated through the autophosphorylation of Threonine 232 residue mainly occurring from the late S-Phase through the M phase during the cellular division cycle or DNA damage response. The overexpression of the Aurora kinases in a wide range of cancers (Leukemia, Colon Cancer, Prostate Cancer, and Breast Cancer) makes them interesting candidates for the development of Aurora kinase inhibitors (AKIs).
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
Phospho-Aurora B (Thr232) assay principle
The Phospho-Aurora B (Thr232) assay measures Aurora B when phosphorylated at Thr232. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Phospho-Aurora B (Thr232) 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, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both 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.

 

Phospho-Aurora B (Thr232) 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 adding Phospho-Aurora B (Thr232) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Phospho-Aurora B (Thr232) 1-plate assay protocol
Detection of Phosphorylated Aurora B (Thr232) 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-500 assay points