Revvity, 64RNAP2S5PEH, HTRF Human and Mouse Phospho-RNA Polymerase II (Ser5) Detection Kit, 10,000 Assay Points

Overview
RNA polymerase II (RNA Pol II) is one of the primary enzymes responsible for expressing protein-encoding genes and some small nuclear RNAs. The carboxy-terminal domain (CTD) of RNA Pol II undergoes a cycle of phosphorylation, catalyzed mainly by members of the cyclin-dependent kinase (CDK) family. RNA Pol II CTD phosphorylation allows it to temporally couple transcription with transcription-associated processes (binding specificity of the CTD for particular factors is determined by the site-specific phosphorylation/dephosphorylation events).
How it works
Phospho-RNA Pol II (Ser5) assay principle
The Phospho-RNA Pol II (Ser5) assay measures RNA Pol II when phosphorylated at Ser5. 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 leads to the formation of an immune-complex 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-RNA Pol II (Ser5) 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 RNA Pol II (Ser5) HTRF detection reagents. This protocol allows the cells' viability and confluence to be monitored.

Phospho-RNA Pol II (Ser5) one-plate assay protocol
Detection of Phosphorylated RNA Pol II (Ser5) 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 allows miniaturization while maintaining robust HTRF quality. Application-Protein Quantification
Brand-HTRF
Buffer/Solvent-Lysis Buffer 1
Detection Modality-HTRF
Host Species-Human Mouse
Molecular Modification-Phosphorylation
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target-RNA pol II
Target Class-Phosphoproteins
Target Species-Human Mouse
Technology-TR-FRET
Therapeutic Area-Immuno-oncology Inflammation
Unit Size-10,000 assay points