HTRF Human & Mouse Total BRD7 Detection Kit, 500 Assay Points

Total-BRD7 assay principle

The HTRF Total-BRD7 assay quantifies the expression level of BRD7 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total-BRD7 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 BRD7 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-BRD7 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-BRD7 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total-BRD7 one-plate assay protocol

Detection of Total-BRD7 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 different immortalized tumoral Human and Mouse cell models

The adherent Immune Human cell lines MCF7 (mammary gland), HELA (cervix), SH-SY5Y (neuroblast from bone narrow), HEK293(embryonic kidney) and the Mouse cell lines NIH3T3 and Neuro2A were plated in 96-well culture plates at a density of 50,000 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 BRD7 expression level was assessed with the HTRF Total BRD7 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 BRD7 assay efficiently detected BRD7 in various cellular models expressing different levels of the protein.

Specificity of HTRF Total BRD7 assay using knockout HAP1 cell lines

BRD7 expression level was assessed with HTRF total BRD7 kit in HAP1 cells (WT) and five HAP1 different cell lines knocked-out for BRD9, BRD7 (Family IV), BRD2, BRD3, or BRD4 (BET Family). 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 BRD7 cells, the HTRF signal iwas equivalent to the non-specific signal (dotted line) indicating a complete BRD7 gene silencing, whereas the BRD7 level is well detected in the other cell lines, as expected. This result demonstrates the selectivity of the HTRF BRD7 kit over BRD9, even though these two proteins share more than 50% sequence homology.

Note that the BRD7 expression level was significantly higher in HAP1 KO BRDs cell lines compared to the WT cell line, which may indicate a compensatory mechanism.

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 BRD7 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 BRD7 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 BRD7 assay was 8-fold more sensitive than the Western Blot technique.