Revvity, 64CYCB1S1PEH, HTRF Human Phospho-Cyclin B1 (Ser126) Detection Kit, 10,000 Assay Points

Overview
This HTRF cell-based assay provides a convenient and accurate way to detect phosphorylated Cyclin B1 at Ser126.
Cyclin B family members (including Cyclin B1 and Cyclin B2) are important regulators of cell cycle progression. They are integral mediators of the growth factor-dependent transition from G2 to Mitosis phases, playing the role of the regulatory subunit of CDK1.
Cyclin B1 is upregulated in the presence of extracellular mitogenic stimuli and is subsequently rapidly downregulated by proteasomal degradation following its phosphorylation on Ser126.
Cyclin B1 is often overexpressed in many cancers. Such abnormal overexpression of Cyclin B1 results in dysregulated CDK1 activity and can lead to tumorigenesis.
How it works
Phospho-Cyclin B1 (Ser126) assay principle
The Phospho-Cyclin B1 (Ser126) assay measures Cyclin B1 when phosphorylated at Ser126. 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. This 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-Cyclin B1 (Ser126) 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-Cyclin B1 (Ser126) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-Cyclin B1 (Ser126) one-plate assay protocol
Detection of Phosphorylated Cyclin B1 (Ser126) 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
Molecular Modification-Phosphorylation
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Phosphoproteins
Technology-TR-FRET
Unit Size-10,000 assay points