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HTRF Human Total BRD4 Detection Kit HTRF®

The Total BRD4 kit is designed to quantify the expression level of BRD4 in cells.

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  • Low sample consumption Low sample consumption
  • All inclusive kit All inclusive kit
  • No-wash No-wash
  • High sensitivity High sensitivity

The Total BRD4 kit is designed to quantify the expression level of BRD4 in cells.

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Overview

BRD4 is a transcriptional regulator involved in embryogenesis and cancer development. Like the other members of the Bromodomains and Extraterminal (BET) family (BRD2, BRD3, and BRDT), BRD4 comprises two bromodomains which bind acetylated lysine residues on proteins, such as histones. Associated with Cyclin T1 and CDK9, the three proteins form a multiprotein complex called P-TEFb, and control gene transcription. As well as its role in transcriptional control, BRD4 is involved in DNA damage response.

Inhibition of BRD4 function is part of the strategy for cancer treatments. More recently, targeting BRD4 degradation via PROTAC molecules has emerged as a novel and promising therapeutic approach.

Benefits

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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

Principle of the HTRF total BRD4 assay

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.

Two-plate protocol of the HTRF total BRD4 assay

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.

One-plate protocol of the HTRF total BRD4 assay

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.

Validation on various cell lines Total BRD4 assay

PROTAC® induced BRD4 degradation

Pharmacological validation Total BRD4 assay-PROTAC on MCF7 cells
Pharmacological validation Total BRD4 assay-GAPDH on MCF7 cells

MCF7 cells were cultured in a 96-well plate (25,000 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 dBET6, ARV-771 ,and ZXH-3-26, 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) was 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 BRD4 detection antibodies were added. The HTRF signal was recorded after an overnight incubation.


The three BRD4 PROTAC degraders trigger a dose-dependent decrease in BRD4 protein with a DC50* in the nM range, as  expected (Raina et al. Proc Natl Acad Sci U. 2019; Winter et al. Mol Cell. 2017; Nowaket al.Nat.Chem.biol. 2018), while the expression level of BRD4 in the ARV-471 control condition remained stable.

A decrease in the housekeeping GAPDH signal was observed in the same conditions, indicating a potential cytotoxicity induced at  high PROTAC concentrations.

* DC50  corresponds to the concentration of the degrader at which 50% of the targeted protein is degraded.

Specificity of HTRF Total BRD4 assay using siRNA

MCF7 cells were plated in 96-well plates at 25,000 cells/well and cultured for 24h. The cells were then transfected with siRNAs specific for BRD1/2/3/4/7/9 and BRDT, as well as with a negative control siRNA. After a 24h incubation, the cells were lyzed 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 BRD4 detection antibodies. The HTRF signal was recorded after an overnight incubation.

BRD4 siRNA led to a 56% BRD4 expression level decrease compared to the negative siRNA, while the HTRF BRD4 signal remained stable in other BRD siRNA transfected cells. Note that the GAPDH housekeeping signal did not change in the same conditions. Taken together, these results demonstrate the specificity and selectivity of the HTRF BRD4 assay.

Specificity of Total BRD4 assay using siRNA – BRD4
Specificity of Total BRD4 assay using siRNA – GAPDH

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.

HTRF total BRD4 assay compared to Western Blot

BRD4 Signaling Pathway

BRD4 binds hyperacetylated chromatin regions, also called super-enhancer regions, and recruits several transcriptional co-activators promoting the assembly of a large platform of transcription regulating proteins. This complex further forms a bridge between Super Enhancer and Promoter where NFKB and other transcription factors (TF) bind. Finally, P-TEFb stimulates RNA-PolII which in turn initiates the transcription of several genes, including oncogenes such as c-Myc or K-Ras.

Scheme adapted from Muddassir, et al RSC Adv., 2021.

HTRF total BRD4 assay signaling pathway

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Plate Reader Requirement

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