Cell-based Epigenetic Assays

Purpose: Quantification of acetylated tubulin as a readout of HDAC activity for drug screening

Targets: HDAC

Assay format: Western Blot

Assay Format: ELISA

Cells incubate with the compound. Subsequently, cells are lysed, and the proteins are solubilized via a detergent. Next, the solution is transferred to an ELISA plate coated with anti-alpha-tubulin antibodies for capturing alpha-tubulin. After washing, an acetyl-lysine antibody is added to bind to acetylated alpha-tubulin. A secondary HRP-linked antibody is used for the quantification of bound detection antibodies which is proportional to the amount of acetylated alpha-tubulin in the sample.  

Purpose: Quantification of acetylated histones as a readout of HDAC activity for drug screening

Targets: HDAC

Assay format: ELISA

Cells incubate with the compound. Subsequently, cells are lysed, and the proteins are solubilized via a detergent. Next, the solution is transferred to an ELISA plate coated with anti-Histone H3 antibodies to capture Histone H3. After washing, an acetyl-lysine antibody is added to bind to acetylated Histone H3. A secondary HRP-linked antibody is used for quantification of bound detection antibodies which is proportional to the amount of acetylated Histone H3 in the sample.  


Assay format: HDAC-Glo
Cells incubate with the compound for 1 hour in serum-free medium. The HDAC-Glo substrate is added with is an acetylated peptide fused to aminoluciferin. After deacetylation of the peptide by HDAC activities, the peptide is cleaved via a protease in the reagent to release aminoluciferin substrate which is quantified in a reaction using luciferase. The luminescence is quantified via an Envision 2104 Multilabel Reader.

Purpose: Quantification of methylated histone as a readout of methyltransferase activity for drug screening

Targets: Methyltransferase

Assay format: Western Blot

Cells incubate with the compound. Subsequently, cells are lysed, and the proteins are solubilized via a detergent. Next, the proteins are separated by their molecular size via SDS-PAGE before being transferred onto a membrane. Tri-methylated Histone H3 is detected by a specific antibody labeled with fluorochromes and scanned. The membrane is then reprobed with an antibody detecting Histone H3 for normalization purposes.

The membrane is scanned with the LICOR Odyssey Infrared imager for quantification of Histone H3 and tri-methylated Histone H3 in the sample.

Purpose: Quantification of phosphorylated histone as a readout of methyltransferase activity for drug screening

Targets: Kinases

Assay format: ELISA

Cells incubate with the compound for 90 minutes in serum-free media. After treatment with phosphatase inhibitors, cells are lysed, and the proteins are solubilized via a detergent. Next, the solution is transferred to an ELISA plate coated with anti-Histone H3 antibodies to capture Histone H3. After washing, a phospho-Histone 3 antibody is added to detect phosphorylated Histone H3. A secondary HRP-linked antibody is used for quantification of bound detection antibodies which is proportional to the amount of phosphorylated Histone H3 in the sample.

Purpose: Testing the efficacy of epigenetic drug candidates to interrupt the interaction of a bromodomain protein with chromatin

Targets: Bromodomains

Assay Format: NanoBRET Bromodomain:Histone Interaction Assay (Promega)

HEK 293T cells are transfected with two constructs: a bromodomain-NanoLuc fusion construct and a Histone-HaloTag fusion construct.

After 24 hours, the Halo Ligand and the test compound will be added. The interaction of bromodomain and histone will allow for electron transfer between the NanoLuc to the HaloLigand eliciting fluorescence in the presence of the NanoLuc substrate.

Purpose: Testing the binding affinity of epigenetic drug candidates to their target in intact cells

Targets: Bromodomains, HDAC

Assay Format: NanoBRET Intracellular Target Engagement Assay (Promega)NanoBRET assays are compound competition assays that rely on bioluminescence resonance energy transfer (BRET) between a luciferase-tagged target and a fluorescent tracer. In the presence of the compound, the tracer is replaced diminishing the fluorescence.

Quantification and specificity are key attributes of the NanoBRET system.