Revvity, 64CRBNPEH, HTRF Human and Mouse Total Cereblon Detection Kit, 10,000 Assay Points

Overview
The Total Cereblon cellular assay measures the expression level of the E3 ubiquitin ligase Cereblon (CRBN) in cells.
Cereblon is a ubiquitously expressed protein which functions as a substrate recognition subunit within the CRL4-CRBN E3 ubiquitin ligase complex, and targets specific proteins for proteolysis through the Ubiquitin Proteasome System (UPS).
Cereblon plays a key role in normal limb outgrowth, normal cell proliferation and metabolism, and in memory and learning.
Cereblon is also the primary direct target of the immunomodulatory drugs (IMiDs) Thalidomide, Pomalidomide, and Lenalidomide, that are all FDA approved drugs for the treatment of multiple myeloma. Interestingly, IMiDs have recently been derived into PROTAC molecules in order to direct degradation of undesired proteins by the UPS.
How it works
Total Cereblon assay principle
The Total Cereblon assay quantifies the expression level of Cereblon in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total Cereblon assay uses two labeled antibodies, one coupled to a donor fluorophore and the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In the presence of Cereblon 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 Cereblon 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 Cereblon HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Total Cereblon one-plate assay protocol
Detection of Total Cereblon 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 4 Lysis Buffer 5
Molecular Modification-Total
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Phosphoproteins
Technology-TR-FRET
Therapeutic Area-Inflammation Neuroscience Oncology & Inflammation
Unit Size-10,000 assay points