Revvity, 64AURATPEH, HTRF Human Total Aurora A Detection Kit, 10,000 Assay Points

Overview
This HTRF cell-based assay conveniently and accurately detects human Aurora A protein (related to the AURKA gene) ubiquitously expressed in eukaryotes. Aurora A belongs to the serine/threonine Aurora kinases family (including Aurora B and Aurora C), essential for cell division via mitosis regulation and especially the process of chromosomal segregation. It is activated through the autophosphorylation of Threonine 288 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).
How it works
Total Aurora A assay principle
The Total-Aurora A assay quantifies the expression level of Aurora A in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total-Aurora A 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 Aurora A 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.

 

Total-Aurora A 2-plate assay protocol
The 2-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring the lysates into a 384-well low volume detection plate before the addition of Total Aurora A HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Total-Aurora A 1-plate assay protocol
Detection of total Aurora A 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-Total
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