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.

Validation on various Human and Mouse cell lines

The adherent Human cell lines MCF7 and MDA-MB-543 cells (mammary gland), HELA (cervix), SH-SY5Y (neuroblast from bone narrow), HEK-293 (embryonic kidney), and the Mouse cell lines NIH3T3 and Neuro2A were plated in 96-well culture plates at a density of 100,000 cells /well and incubated for 24 hours at 37°C, 5% CO2. After culture medium removal, the cells were lysed with 50 µL of supplemented lysis buffer #4 (1X).

The suspension immune Human PL-21 cell line (Acute myeloid Leukemia) was dispensed under 30 µL in a 96-well plate at a density of 100,000 cells/well, incubated for 1h at 37°C-5% CO2, and lysed with 10 µL of supplemented lysis buffer #4 (4X).

The BRD4 expression level was assessed with the HTRF Total BRD4 kit. Briefly, 16 µL of cell lysate were transferred into a low volume white microplate, followed by 4 µL of premixed HTRF detection reagents. The HTRF signal was recorded after an overnight incubation at RT. The dotted line corresponds to the non-specific HTRF signal. Note that the cell density was previously optimized to ensure HTRF detection within the dynamic range of the kit (data not shown).

The HTRF Total BRD4 assay efficiently detects BRD4 in various cellular models expressing different levels of the protein.

HTRF total BRD4 assay compared to Western Blot

MCF7 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) after cell medium removal, for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer#4, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF total BRD4 detection reagents.

Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

In these conditions, the HTRF total BRD4 assay was 8-fold more sensitive than the Western Blot technique.