HTRF Human Total BRD7 Detection Kit HTRF®
The Human BRD7 kit is designed to monitor the expression level of cellular BRD7 protein.
- Low sample consumption
- All inclusive kit
- High sensitivity
The Total BRD7 cellular assay monitors BRD7 protein levels.
Bromodomain containing 7 (BRD7), also known as NAG4, BP75 or CELTIX1, is a member of the bromodomain-containing protein family IV, which also includes BRD9 protein. BRD7 is mainly localized in the nucleus and regulates chromatin remodeling. BRD7 is a component of a large polybromo-BAF (PBAF) complex, including ARID2, PBRM1, and BAF45d, which interacts with transcriptional regulators to modulate gene expression.
More importantly, recent studies have shown that BRD7 is downregulated in certain cancers such as epithelial ovarian carcinoma, breast cancer, nasopharyngeal cancer, and colorectal carcinoma. Unlike prooncogenic BRD9 roles, BRD7 may function as a tumor suppressor by inhibiting the Phosphoinositide 3-kinase (PI3K) pathway involved in various deregulated oncogenic processes, such as apoptosis, protein synthesis, proliferation, and angiogenesis.
Since BRD7 and BRD9 proteins share a 72% residue similarity, compounds are likely to target both the tumor suppressor BRD7 and the prooncogene BRD9. Therefore, the selectivity of compounds targeting either BRD9 or BRD7 must be carefully investigated. To do this, the HTRF total BRD7 kit is a valuable companion assay for the HTRF total BRD9 kit, enabling reliable compound profiling.
- COMPOUND PROFILING
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
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.
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.
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.
MCF7 and HeLa cells were cultured in a 96-well plate (50,000 cells/well) for 24 hours at 37°C, 5% CO2. After cell culture medium removal, they were treated with increasing concentrations of the PROTAC compounds dBRD9 and VZ185, as well as a PROTAC ARV-471 (PROTAC Estrogen Receptor) used as a negative irrelevant control. After overnight incubation, the cell culture medium was removed and 50 µl of supplemented Lysis Buffer#4 (1X) were dispensed into each well. After cell lysis, 16 µL of lysates were transferred into a 384-well low volume white microplate and 4 µL of the HTRF BRD7 detection antibodies were added. The HTRF signal was recorded after an overnight incubation.
In both cell types, only VZ185 degrader triggered a dose-dependent decrease in BRD7 protein, while the expression level of BRD7 in BRD9 selective PROTAC (dBRD9) and ARV-471 control condition. Note that unlike the ARV-471 treated MCF7 cells, GAPDH remained stable in HeLa cells.
These results suggest a VZ185 PROTAC induced BRD7 degradation, with a DC50* in the nM range, as reported by Zoppi et al (J.Med.Chem. 2019)
* DC50 corresponds to the concentration of the degrader at which 50% of the targeted protein is degraded.
Since BRD9 is an oncogene and BRD7 plays a tumor suppressor role, the selective degradation of BRD9 rather than BRD7 is of major importance.
HeLa cells 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 and VZ185 PROTAC compounds. After overnight treatment, the cell culture medium was removed and 50 µl of supplemented Lysis Buffer#4 (1X) were dispensed into each well. After cell lysis, 16 µL of lysates were transferred into a 384-well low volume white microplate and 4 µL of the HTRF detection antibodies were added using Total BRD9 or Total BRD7 assay reagents. The HTRF signal was recorded after an overnight incubation.
These results indicate a VZ185 induced dose-dependent BRD9 and BRD7 decrease, whereas dBRD9 induces a dose-dependent decrease in BRD9 only. The GAPDH remained stable in the same conditions (data not shown). This result demonstrates a dBRD9 mediated selective BRD9 degradation, as described by Remillard et al. (Angew Chem Int Ed Engl. 2017) and Zoppi et al.(J.Med.Chem.; 2019).
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.
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.
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