Custom Cellular Epigenetic Assays
Readout | Assay format |
---|---|
Histone & Tubulin Deacetylation | Western Blot; ELISA |
Histone Deacetylation (Class I/II) | HDAC-Glo |
Histone Deacetylation (Class IIa) | HDAC-Glo |
Histone Methylation | Western Blot |
Histone Phosphorylation | ELISA |
Target-specific Cellular HDAC and Bromodomain Assays
Target | HGNC Reference | Additional Synonyms | Assay Format | Data sheet |
---|---|---|---|---|
BRD2* | BRD2 | KIAA9001; RING3; D6S113E; NAT; FSRG1 | NanoBRET Target Engagement | Please inquire |
BRD2-1* | BRD2 (BD1) | KIAA9001; RING3; D6S113E; NAT; FSRG1 | NanoBRET Target Engagement | Please inquire |
BRD2-2* | BRD2 (BD2) | KIAA9001; RING3; D6S113E; NAT; FSRG1 | NanoBRET Target Engagement | Please inquire |
BRD3* | BRD3 | RING3L; ORFX; KIAA0043 | NanoBRET Target Engagement | Please inquire |
BRD4 | BRD4 | HUNKI; MCAP; CAP; HUNK1 | NanoBRET Bromodomain Histone Interaction | Data sheet |
BRD4 | BRD4 | HUNKI; MCAP; CAP; HUNK1 | NanoBRET Target Engagement | Data sheet |
BRD4-1* | BRD4 (BD1) | HUNKI; MCAP; CAP; HUNK1 | NanoBRET Target Engagement | Please inquire |
BRD4-2 | BRD4 (BD2) | HUNKI; MCAP; CAP; HUNK1 | NanoBRET Target Engagement | Data sheet |
BRDT* | BRDT | BRD6; CT9 | NanoBRET Target Engagement | Please inquire |
CBP | CREBBP | CBP-BD, CREB binding protein, RSTS, RTS, CBP, KAT3A | NanoBRET Bromodomain Histone Interaction | Data sheet |
HDAC1* | HDAC1 | HDAC 1; HD1; GON-10; KDAC1; RPD3L1 | NanoBRET Target Engagement | Please inquire |
HDAC10 | HDAC10 | HDAC 10; DKFZP761B039 | NanoBRET Target Engagement | Data sheet |
HDAC2* | HDAC2 | HDAC 2; RPD3; YAF1; KDAC2 | NanoBRET Target Engagement | Please inquire |
HDAC3* | HDAC3 | HDAC 3; RPD3; HD3; RPD3-2; SMAP45; KDAC3 | NanoBRET Target Engagement | Please inquire |
HDAC6 | HDAC6 | HDAC 6; KIAA0901; JM21; HD6; FLJ16239; PPP1R90 | NanoBRET Target Engagement | Data sheet |
HDAC6-2 | HDAC6 (CD2) | HDAC 6; KIAA0901; JM21; HD6; FLJ16239; PPP1R90 | NanoBRET Target Engagement | Data sheet |
* Please allow 2 weeks for final assay establishment at the Reaction Biology screening facility before the client compound testing begins.
Cell-based Epigenetic Assay Details
Purpose: Quantification of acetylated tubulin as a readout of HDAC activity for drug screening
Targets: HDAC
Assay format: Western Blot
The membrane is scanned with the LICOR Odyssey Infrared imager for quantification of alpha-tubulin and acetylated alpha-tubulin in the sample.
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.