Bromodomain-containing protein 4 (BRD4) is a member of the Bromodomains and Extraterminal (BET) family, consisting of BRD2, BRD3, and BRDT. BRD4 is a transcription regulator consisting of two bromodomains which bind acetylated lysine residues on proteins, such as histones. In association with Cyclin T1 and CDK9, BRD4 forms a complex called P-TEFb which controls gene transcription. BRD4 is also involved in DNA damage response, embryogenesis, and cancer development. Inhibiting BRD4 is a strategy for treating cancer, and more recently targeting BRD4 with PROTAC molecules has emerged as a novel therapeutic approach.
The AlphaLISA SureFire Human Total BRD4 Detection Kit is a sandwich immunoassay for the quantitative detection of total BRD4 in cellular lysates, using Alpha Technology.
Formats
The HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume.
The 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume.
The 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume.
The 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume.
AlphaLISA SureFire Ultra kits are compatible with
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
Alpha SureFire kits can be used for
Cellular kinase assays
Receptor activation studies
Screening
Total-AlphaLISA SureFire Ultra assay principle
The Total-AlphaLISA SureFire Ultra assay measures the expression level of a target protein in a biological sample (e.g. cell lysate).
The Total-AlphaLISA SureFire Ultra assay uses two antibodies which recognize two different distal epitopes on the target protein. AlphaLISA assays require two bead types: Acceptor and Donor Beads. Acceptor Beads are coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody, labeled with a CaptSure tag. Donor Beads are coated with streptavidin to capture one of the detection antibodies, which is biotinylated. In the presence of target protein, the two antibodies bring the Donor and Acceptor Beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor Bead, allowing for the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of protein present in the sample.
Total-AlphaLISA SureFire Ultra two-plate assay protocol
The two-plate protocol involves culturing and treating the cells in a 96-well plate before lysis, then transferring lysates into a 384-well OptiPlate™ plate before the addition of Total-AlphaLISA SureFire Ultra detection reagents. This protocol enables cell viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.
Total-AlphaLISA SureFire Ultra one-plate assay protocol
Detection of Total target protein with AlphaLISA SureFire Ultra reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol allows for miniaturization while maintaining robust AlphaLISA SureFire Ultra quality.
PROTAC degradation of BRD4 in PBMCs
PBMCs were isolated from healthy donors using Ficoll® Plaque Plus and seeded in a 96-well plate (400,000 cells/well) in complete DMEM. Cells were treated with increasing concentrations of ARV-771 for 20 hours.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). BRD4 and Cofilin levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 40,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, ARV-771 triggered a dose-dependent decrease in the levels of BRD4 while Cofilin levels remained unchanged.
PROTAC degradation of BRD4 in endogenous cell systems
HeLa cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated for 24 hours at 37°C, 5% CO2. Cells were treated with increasing concentrations of ARV-771 for 18 hours.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). BRD4 levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate (approximately 4,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, ARV-771 triggered a dose-dependent decrease in the levels of BRD4.
THP1 cells were seeded in a 96-well plate (500,000 cells/well) in HBSS + 0.1% BSA. Cells were treated with increasing concentrations of MZ-1 for 4 hours.
After treatment, cells were lysed with the addition of 50 µL of 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). BRD4 and CDK9 levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 20,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, MZ-1 triggered a dose-dependent decrease in the levels of BRD4, while CDK9 levels were unchanged.
