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.

Assessment of Cereblon protein levels in various human and mouse cell lines

The human HEK293, HepG2, HeLa, and Jurkat cell lines, as well as the mouse NIH-3T3 cell line, were seeded in 96-well plates at different cell densities and cultured for 24h. The cells were then lysed with supplemented lysis buffer #4, and the cell lysates were analyzed for their total Cereblon protein levels by transferring 16 µL of samples into a 384-well low volume white microplate and adding 4 µL of the HTRF Total Cereblon detection antibodies. The HTRF signal was recorded after an overnight incubation.

Knockdown of CRBN expression by siRNA experiments

HEK293 cells were plated in a 96-well plate (50,000 cells/well) and cultured for 24h. The cells were then transfected either with a siRNA specific for Cereblon or with a siRNA specific for VHL (Von Hippel–Lindau), as well as with a negative control siRNA. After a 48h incubation, the cells were lysed and 16 µL of lysates were transferred into a 384-well low volume white microplate, before the addition of 4 µL of the HTRF Total Cereblon detection antibodies. The HTRF signal was recorded after an overnight incubation.

As expected, cell transfection with the Cereblon siRNA induced the knockdown of CRBN expression as highlighted by the 76% signal decrease, while no signal modulation was observed when cells were transfected with the VHL siRNA or with the negative siRNA.

Stabilization of Cereblon protein levels by immunomodulatory drugs (IMiDs)

HEK293 cells were seeded in a 96-well plate (50,000 cells/well) and incubated for 6h. The cells were then treated with 0.1 and 10 µM of the IMiDs Thalidomide (TOCRIS, #0652), Lenalidomide (TOCRIS, #6305), and Pomalidomide (TOCRIS, #6302). After an overnight incubation, the cells were lysed and 16 µL of lysates were transferred into a 384-well low volume white microplate, before the addition of 4 µL of the HTRF Total Cereblon detection antibodies. The HTRF signal was recorded after an overnight incubation.

As expected, cell treatment with Thalidomide, andwith its derivatives Lenalidomide and Pomalidomide, resulted in stabilization and accumulation of Cereblon protein levels through the inhibition of Cereblon ubiquitination and degradation (Liu et al., A novel effect of thalidomide and its analogs: suppression of cereblon ubiquitination enhances ubiquitin ligase function; FASEB J. 2015 Dec; 29(12): 4829–4839).

HTRF total Cereblon assay compared to Western Blot

HEK293 cells were cultured in a T175 flask in complete culture medium at 37°C-5%, CO2. After a 48h incubation, the cells were lysed with 3 mL of supplemented lysis buffer #4 (1X) for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF total Cereblon detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.

Using the HTRF total Cereblon assay, only 500 cells/well were enough to detect a significant signal, while 4,000 cells/track were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore in these conditions, the HTRF Total Cereblon assay was 8 times more sensitive than the Western Blot technique.