Revvity, 64AT5TPEG, HTRF Human and Mouse Total STAT5 Detection Kit, 500 Assay Points

Overview
Signal Transducer and Activator of Transcription (STAT) proteins are a seven-member family of cytoplasmic transcription factors that relay signals emanating from cell-surface cytokine and growth factor receptors to the nucleus. STAT proteins control fundamental cellular processes, including cell survival, proliferation, differentiation, and immune responses. Dysregulation of STAT5 signaling is implicated in hematopoietic cancers, where it promotes cell survival, proliferation, and metastasis.
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
Total STAT5 assay principle
The Total STAT5 assay quantifies the expression level of STAT5 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total STAT5 assay uses two labeled antibodies, one coupled to a donor fluorophore and the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In the presence of STAT5 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 STAT5 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 HTRF Total STAT5 detection reagents. This protocol allows for the cells' viability and confluence to be monitored.

Total STAT5 one-plate assay protocol
Detection of Total STAT5 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 facilitates miniaturization while maintaining robust HTRF quality. Application-Cell Signaling
Brand-HTRF
Buffer/Solvent-Lysis Buffer 4
Detection Modality-HTRF
Host Species-Human Mouse
Molecular Modification-Total
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target-STAT5
Target Class-Phosphoproteins
Technology-TR-FRET
Therapeutic Area-Inflammation Oncology
Unit Size-500 assay points