The BRD4 (BD2) Inhibitor Screening Assay Kit is designed to measure the inhibition of BRD4 bromodomain 2 (BD2) binding to its substrate. The key to the BRD4 (BD2) Inhibitor Screening Assay Kit is the highly specific binding of the BRD4 bromodomain 2 to the acetylated histone substrate. With this kit, only three simple steps on a microtiter plate are required. First, a sample containing BRD4 bromodomain 2 and an inhibitor of choice is incubated with the biotinylated substrate for thirty minutes. Next, acceptor beads are added, then donor beads, followed by reading the Alpha-counts.

MATERIALS OR INSTRUMENTS REQUIRED BUT NOT SUPPLIED:
Glutathione AlphaLISA^® Acceptor Beads, 5 mg/ml (PerkinElmer #AL109C)
AlphaScreen^® Streptavidin-conjugated donor beads, 5 mg/ml (PerkinElmer #6760002)
Optiplate-384 (PerkinElmer #6007290)
AlphaScreen^® microplate reader
Adjustable micropipettor and sterile tips

BRD4, Bromodomain, HUNK1, MCAP, activity assay kit

Kit comes in a convenient AlphaLISA^® format, with biotinylated histone peptide substrate, assay buffer, detection buffer and purified, GST-tagged BRD4 BD2 to perform a total of 384 enzyme reactions.

DMSO above 0.5%. Only limited amounts of DMSO can be included, as it has been shown to disrupt BRD-ligand interaction. Avoid green and blue dyes that absorb light in the Alphascreen^® signal emission range (520-620 nm), such as Trypan Blue. Avoid the use of the potent singlet oxygen quenchers such as sodium azide (NaN₃) or metal ions (Fe²⁺, Fe³⁺, Cu²⁺, Zn²⁺ and Ni²⁺). The presence of >1% RPMI 1640 culture medium leads to a signal reduction due to the presence of excess biotin and iron in this medium. MEM, which lacks these components, does not affect Alphascreen^® assays.

COMPONENTS:

See assay kit data sheet for detailed protocol.

At least 12 months from date of receipt, when stored as directed. Kit components require different storage conditions. Be sure to store each component at the proper temperature upon arrival.

Useful for the study of bromodomain binding assays, screening bromodomain inhibitors and selectivity profiling.

1. McBride, A.A., et al., Trends Microbiol. 2004; 12(12):527.

Application Reference(s): 
1. Discovery of a small-molecule bromodomain-containing protein 4 (BRD4) inhibitor that induces AMP-activated protein kinase-modulated autophagy-associated cell death in breast cancer (2017)

MATERIALS OR INSTRUMENTS REQUIRED BUT NOT SUPPLIED:
Glutathione AlphaLISA^® Acceptor Beads, 5 mg/ml (PerkinElmer #AL109C)
AlphaScreen^® Streptavidin-conjugated donor beads, 5 mg/ml (PerkinElmer #6760002)
Optiplate-384 (PerkinElmer #6007290)
AlphaScreen^® microplate reader
Adjustable micropipettor and sterile tips

Avoid freeze/thaw cycles.

Bromodomain

Assay Kit