Revvity, 64NPMT1PEH, HTRF Human and Mouse Phospho-NPM1 (Thr199) Detection Kit, 10,000 Assay Points

Overview
NPM1 (Nucleophosmin 1) is a molecular chaperone involved in cell cycle regulation, ribosome biogenesis, cell proliferation, DNA damage repair, and apoptosis. Its phosphorylation on Thr199 by the CDK2/Cyclin E complex regulates mitosis, centrosome duplication, pre-mRNA processing, and G2/M cell cycle arrest through its interaction with p53/MDM2.
NPM1 functions both as an oncogene and a tumor suppressor. It is frequently overexpressed, altered, and rearranged, or sporadically deleted in human cancers. NPM1 is also the most frequently mutated gene (NPM1c) in acute myeloid leukemia (AML).
How it works
Phospho-NPM1 (Thr199) assay principle

The Phospho-NPM1 (Thr199) assay measures NPM1 when phosphorylated at Thr199. 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 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-NPM1 (Thr199) 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-NPM1 (Thr199) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 
Phospho-NPM1 (Thr199) one-plate assay protocol

Detection of Phosphorylated NPM1 (Thr199) 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-Cell Signaling
Automation Compatible-Yes
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 4
Molecular Modification-Phosphorylation
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target-NPM1
Target Class-Phosphoproteins
Target Species-Human Mouse
Technology-TR-FRET
Therapeutic Area-Oncology
Unit Size-10,000 assay points