Total-BRD9 assay principle

The HTRF Total-BRD9 assay quantifies the expression level of BRD9 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 the presence of BRD9 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-BRD9 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-BRD9 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total-BRD9 one-plate assay protocol

Detection of Total-BRD9 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), and 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 BRD9 expression level was assessed with the HTRF Total BRD9 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 BRD9 assay efficiently detects BRD9 in various cellular models expressing different levels of the protein.

Specificity of HTRF Total BRD9 assay using knockout HAP1 cell lines

BRD9 expression level was assessed with the HTRF total BRD9 kit in HAP1 cells (WT) and five HAP1 different cell lines knocked-out for BRD9, BRD7 (Family IV), BRD2, BRD3 or BRD4 (BET Family). The cell density was previously optimized to ensure HTRF detection within the dynamic range of the kit (data not shown).

The 6 different cell lines were cultured in a 96-well plate (50,000 cells/well) 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), then 16 µL of cell lysate were transferred into a low volume white microplate followed by 4 µL of premixed detection reagents. The HTRF signal was recorded after an overnight incubation at RT.

In HAP1 KO BRD9 cells, the HTRF signal was equivalent to the non-specific signal (dotted line) indicating a complete BRD9 gene silencing, whereas the BRD9 level was well detected in the other cell lines, as expected.

Note that i) the BRD9 expression level was significantly higher in HAP1 KO BRD3 cell line, suggesting a compensatory mechanism, ii) Similar expression levels of BRD9 in WT and KO BRD7 cell lines demonstrate the selectivity of the HTRF BRD9 kit over BRD7, as these two proteins share more than 50% sequence homology.

Catalog cell line references (Horizon Discovery) : HAP1 Wt #C631; HAP1 KO BRD9 #HZGHC000934c003; HAP1 KO BRD7 #HZGHC000923c010; HAP1 KO BRD2 # HZGHC000356c015; HAP1 KO BRD3 # HZGHC000244c004; HAP1 KO BRD4 # HZGHC000937c007.

HTRF total BRD9 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 BRD9 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 BRD9 assay was 8-fold more sensitive than the Western Blot technique.

dBRD9 PROTAC pharmacology in HTRF and Western Blot

MCF7 were cultured in a 96-well plate (50,000 cells/well) for 24 hours at 37°C, 5% CO2. After cell culture medium removal, cells were treated with increasing concentrations of dBRD9. After overnight treatment, the cell culture medium was removed and 50 µl of supplemented Lysis Buffer#4 (1X) were added to each well. 16µL of the same cell lysates were used to perform Total HTRF BRD9 assay and Western Blot detection using their respective protocols.

Both techniques showed a dBRD9 induces a dose-dependent BRD9 decrease and correlated DC50 (40 nM for HTRF and 90 nM for Western Blot), whereas the GAPDH expression level remained stable (data not shown).