Revvity, 64HSPS78PEG, HTRF Phospho-HSP27 (Ser78) Detection Kit, 500 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
Phospho-HSP27 (Ser78) assay principle
The Phospho-HSP27 (Ser78) assay measures HSP27 when phosphorylated at Ser78. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses 2 antibodies, one labeled with a donor fluorophore and 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 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-HSP27 (Ser78) 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 Phospho-HSP27 (Ser78) HTRF detection reagents. This protocol allows for the cells' viability and confluence to be monitored.

Phospho-HSP27 (Ser78) one-plate assay protocol
Detection of Phosphorylated HSP (Ser78) 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-Phosphorylation
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-500 assay points