Revvity, 64HSPTPEH, HTRF Human Total HSP27 Detection Kit, 10,000 Assay Points

Overview
Heat shock protein (HSP) 27 (also known as HSPB1) is an ATP-independent, small-sized (approximately 27 kDalton) chaperone molecule. HSP27 is present at basal levels in cells and tissues and is organized as large oligomers up to 1,000 kD. Following phosphorylation (Ser-15, Ser-78, Ser-82, Thr-143), HSP27 reorganizes itself into smaller oligomers, often dimers and tetramers and can interact with other proteins. This simple change in phosphorylation state regulates many of the canonical functions of HSP27. HSP27 is reported to be involved in cell resistance to heat shock and several stress factors, including hypoxia, oxidative stress, infections, and ultraviolet radiation. However, the function of HSP27 is hijacked during disease, and HSP27 helps to promote disease, rather than appropriately regulate cell homeostasis. So, HSP27 is overexpressed in various cancer cell lines and tumor progression, metastatic potential and treatment resistance have been attributed to the increased HSP27 expression or HSP27 phosphorylation, making HSP27, a therapeutic target of interest.
How it works
Total HSP27 assay principle
The Total HSP27  assay quantifies the expression level of HSP27 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total HSP27 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 HSP27 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 HSP27 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 HSP27 detection reagents. This protocol allows for the cells' viability and confluence to be monitored.

Total HSP27 one-plate assay protocol
Detection of Total HSP27 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 1
Detection Modality-HTRF
Host Species-Human
Molecular Modification-Total
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target-HSP27
Target Class-Phosphoproteins
Technology-TR-FRET
Therapeutic Area-Inflammation Oncology
Unit Size-10,000 assay points