Revvity, 64BRD4TPEG, HTRF Human and Mouse Total BRD4 Detection Kit, 500 Assay Points

Overview
BRD4 is a transcriptional regulator involved in embryogenesis and cancer development. Like the other members of the Bromodomains and Extraterminal (BET) family (BRD2, BRD3, and BRDT), BRD4 comprises two bromodomains which bind acetylated lysine residues on proteins, such as histones. Associated with Cyclin T1 and CDK9, the three proteins form a multiprotein complex called P-TEFb, and control gene transcription. As well as its role in transcriptional control, BRD4 is involved in DNA damage response.
Inhibition of BRD4 function is part of the strategy for cancer treatments. More recently, targeting BRD4 degradation via PROTAC molecules has emerged as a novel and promising therapeutic approach.
HTRF assays offer many advantages over other technologies:

Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
How it works
Total-BRD4 assay principle
The HTRF Total-BRD4 assay quantifies the expression level of BRD4 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total-BRD4 assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of BRD4 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor, and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format

 

Total-BRD4 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 Total-BRD4 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Total-BRD4 one-plate assay protocol
Detection of Total-BRD4 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
Automation Compatible-Yes
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 1 Lysis Buffer 2 Lysis Buffer 3 Lysis Buffer 4
Molecular Modification-Total
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Phosphoproteins
Target Species-Human Mouse
Technology-TR-FRET
Therapeutic Area-Oncology & Inflammation
Unit Size-500 assay points