Revvity, 64NCLT84PEH, HTRF Human Phospho-Nucleolin (Thr84) Detection Kit, 10,000 Assay Points

Overview
Nucleolin is protein mostly localized in the nucleus, where it is involved in ribosome biogenesis by binding to and stabilizing pre-RNA. It also plays several other roles in conjunction with ribosomes, such as RNA transcription regulation via transcription factor binding and nucleus-cytosol mRNA trafficking. The phosphorylation state of nucleolin regulates its activity by increasing its binding affinity for RNA, and is especially enhanced in response to cellular stress and DNA damage. Abnormal phosphorylation of nucleolin is recognized as a potential source of lack of regulation in mRNA transcription, which can alter the expression of pro- and anti-oncogenic genes and contribute to cancer development or progression.
How it works
Phospho-Nucleolin (Thr84) assay principle
The phospho-Nucleolin (Thr84) assay measures Nucleolin levels in cells. 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. In the presence of Nucleolin, the labeled antibodies form an immune complex, bringing the donor fluorophore into close proximity to the acceptor and generating a FRET signal. The signal intensity is directly proportional to the concentration of total protein present in the sample.

phospho-Nucleolin (Thr84) 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-Nucleolin (Thr84) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

phospho-Nucleolin (Thr84) one-plate assay protocol
Detection of Phospho-Nucleolin (Thr84) 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 2
Molecular Modification-Phosphorylation
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target-Nucleolin
Target Class-Phosphoproteins
Target Species-Human Mouse
Technology-TR-FRET
Therapeutic Area-Inflammation Oncology
Unit Size-10,000 assay points