Revvity, 64CREBBPTPEH, HTRF Human and Mouse Total CREBBP Detection Kit, 10,000 Assay Points

Overview
The HTRF Total CREBBP cell-based assay conveniently and accurately detects the levels of Total CREBBP within cells. This kit is compatible with the buffers from the Total p300 kit, so the same lysate can be used for analyses of both family members.
CREBBP (CREB Binding Protein, also known as CBP or KAT3A) and its paralog p300 (also called EP300 or KAT3B) are key transcriptional co-activators. These Lysine Acetyl Transferases (KATs) form the two-member KAT3 family. They control gene transcription by facilitating the assembly of transcriptional machinery and by acetylating specific lysine residues on core histones and on transcription coregulators. CREBBP and p300 act as network "hubs", with more than 400 proteins to regulate the expression of genes controlling several basic cellular processes, such as proliferation and homeostasis.
Mutations or chromosomal translocations involving CREBBP/p300 lead to gene dysregulation and can result in a variety of human diseases such as cancer, neurodegeneration, and inflammatory diseases. CREBBP/p300 PROTAC degraders have recently been developed for the treatment of human cancers.
How it works
Total CREBBP assay principle
The HTRF Total CREBBP assay quantifies the expression level of CREBBP in cell lysate. Unlike Western Blot, the assay is entirely plate-based, and does not require gels, electrophoresis, or transfer. The Total CREBBP 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 CREBBP 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 CREBBP two-plate assay protocol
The 2-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 the Total CREBBP HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Total CREBBP one-plate assay protocol
Detection of total CREBBP 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
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
Unit Size-10,000 assay points