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Table 1 Screening compatible epigenetic assays

From: Epigenetic assays for chemical biology and drug discovery

Enzyme Assay format Key features of the assay References
Histone deacetylase
(HDAC)
Chemiluminescent (AlphaLISA®) • Assay reported in literature and commercial validated assay kit
• Substrate: histone proteins
• Detection: H3-K9(Ac) or H3-K27(Ac)
• High sensitivity
• High throughput functional assay
122–124
  Chromatin immunoprecipitation • Assay reported in literature using specific commercial reagents
• Detection: Ac-H3
• High sensitivity
• Low throughput assay
125
  Colorimetric (Color de Lys®) • Commercial validated assay kit
• Substrate: peptide containing ε-acetylated lysine
• Detection: deacetylated peptide via coupled assay
• Low sensitivity
• Low/Medium throughput functional assay
• Prone to optical interference with compounds
126
  Fluorometric (Fluor de Lys®) • Assay reported in literature and commercial validated assay kit
• Substrate: peptide containing ε-acetylated lysine
• Detection: deacetylated peptide via coupled assay
• Medium/High sensitivity
• Medium/High throughput functional assay
127, 128
  Luminescence (HDAC-Glo™ I/II) • Assay reported in literature and commercial validated assay kit
• Substrate: peptide containing ε-acetylated lysine
• Detection: deacetylated peptide via coupled assay
• High sensitivity
• Medium/High throughput functional assay
131, 132
  TR-FRET (LANCE® Ultra) • Uses specific commercial reagents
• Substrate: biotinylated Histone H3-K27(Ac) or Histone H3-K9(Ac) peptide
• Detection: H3-K9(Ac) or H3-K27(Ac)
• High sensitivity
• High throughput functional assay
133
  TR-FRET (LanthaScreen™) • Assay reported in literature and commercial validated assay kit
• Ligand: Alexa Fluor® 647-labelled HDAC inhibitor as a tracer
• Detection: displacement of Alexa Fluor® 647-labelled HDAC inhibitor
• High sensitivity
• High throughput binding assay
134
Demethylase (LSD and Jumonji C domain-containing histone demethylase) Colorimetric • Assay reported in literature using specific commercial reagents
• Substrates: Histone H3-K4 peptide
• Detection: H2O2 or H3-K4 via coupled assay
• Low sensitivity
• Low throughput functional assay
• Prone to optical interference with compounds
139–142
  Colorimetric (Epigenase™) • Commercial validated assay kit
• Substrates: Histone H3-K4(Me2) or dimethylated Histone H3-K4 peptide
• Detection: H2O2 via coupled assay
• Low sensitivity
• Low throughput functional assay
• Requires wash steps
• Prone to optical interference with compounds
143
  Fluorescence polarization • Assay reported in literature using methylstatfluor tracer
• Ligand: methylstatfluor tracer
• Detection: displacement of methylstatfluor tracer
• High sensitivity
• High throughput binding assay
144–145
  Fluorometric • Commercial validated assay kit
• Substrate: Histone H3-K4(Me2) peptide
• Detection: H2O2 via coupled assay
• Low sensitivity
• Low throughput functional assay
• Requires wash steps
• Prone to optical interference with compounds
146–147
  Fluorometric • Commercial validated assay kit
• Substrate: Histone H3-K4(Me2) protein
• Detection: formaldehyde via coupled assay
• Low sensitivity
• Low throughput functional assay
• Requires wash steps
• Prone to optical interference with compounds
148–149
  High content screening • Assay reported in literature using specific commercial reagents
• Substrate: Histone H3-K27(Me)3 peptide
• Detection: H3-K27(Me)3
• Medium sensitivity
• Medium throughput functional assay
150
  Mass spectrometry • Assay reported in literature using specific commercial reagents
• Substrate: dimethylated peptides
• Detection: demethylated products
• Medium/High sensitivity
• Low throughput functional assay
• No optical interference from compounds
151, 152
  Radioactive • Assay reported in literature using specific commercial reagents
• Substrate: 3H-labelled methylated histone
• Detection: 3H-formaldehyde via coupled functional assay
• No optical interference from compounds
• Radioactive waste
153, 154
  TR-FRET (LANCE® Ultra) • Commercial validated assay kit
• Substrate: biotinylated Histone H3-K4(Me) peptide
• Detection: H3-K4
• High sensitivity
• High throughput functional assay
155
Histone methyltransferase (HMT) Chemiluminescent (AlphaLISA®) • Commercial validated assay kit
• Substrate: Histone H3-K79(Me2) protein
• Detection: H3-K79(Me2)
• High sensitivity
• High throughput functional assay
161
  Fluorescence polarization • Assay reported in literature using specific commercial reagents
• Substrate: protein or peptide
• Detection: displacement of labelled-AMP in coupled assay
• High sensitivity
• High throughput binding assay
162
  Fluorometric • Assay reported in literature using specific commercial reagents
• Substrate: Histone H3 peptide
• Detection: homocysteine via coupled assay
• High sensitivity
• High throughput functional assay
163–165
  High content screening • Assay reported in literature using specific commercial reagents
• Substrate: Histone H3-K27(Me)3
• Detection: H3-K27(Me)3
• Medium sensitivity
• Medium throughput functional assay
166
  Luminescence • Assay reported in literature using specific commercial reagents
• Substrate: protein or peptide
• Detection: complex coupled assay
• High sensitivity
• High throughput functional assay
167
  Radiometric • Assay reported in literature using specific commercial reagents
• Substrate: biotinylated Histone H3-K9 peptide
• Detection: 3H-incorporated into peptide
• No optical interference from compounds
• Radioactive waste
168–170
Histone acetyltransferase (HAT) Colorimetric • Commercial validated assay kit
• Substrate: proprietary peptide
• Detection: NADH via coupled assay
• Low sensitivity
• Low/Medium throughput functional assay
• Prone to optical interference with compounds
174
  ELISA • Commercial validated assay kit
• Substrate: histone
• Detection: H3-K4(Ac) via coupled assay
• Low/Medium sensitivity
• Requires wash steps
• Medium throughput functional assay
175
  Fluorometric • Assay reported in literature using specific commercial reagents
• Substrate: Histone H3 or Histone H4 peptide
• Detection: CoA-SH via coupled assay
• High sensitivity
• High throughput functional assay
• Prone to optical interference with compounds
176
  Fluorometric • Commercial validated assay kit
• Substrate: Histone protein
• Detection: CoA-SH via coupled assay
• High sensitivity
• High throughput functional assay
177
  Microfluidic mobility shift • Assay reported in literature using specific commercial reagents
• Substrate: fluorolabelled Histone H3 or Histone H4 peptide
• Detection: charge difference of substrate/product
• High sensitivity
• Medium throughput functional assay
• No optical interference from compounds
178
  Radiometric • Assay reported in literature using specific commercial reagents
• Substrate: biotinylated Histone H4 peptide or histone protein
• Detection: 3H-incorporated into peptide or histone
• High sensitivity
• Low throughput functional assay
• No optical interference from compounds
• Radioactive waste
179–181
  TR-FRET(LANCE® Ultra) • Commercial validated assay kit
• Substrate: biotinylated Histone H3-K9 peptide
• Detection: H3-K9(Ac)
• High sensitivity
• High throughput binding assay
182
Bromodomain Chemiluminescent (AlphaScreen™) • Assay reported in literature using specific commercial reagents
• Substrate: biotinylated Histone H4-K5(Ac)
• Detection: presence of BRD4/peptide complex
• High sensitivity
• High throughput binding assay
188, 189
  Differential Scanning Fluorometry (BromoMELT™) • Assay reported in literature and commercial validated assay kit
• Substrate: BRD4
• Detection: T m of BRD4/SYPRO Orange complex
• Low/Medium sensitivity
• Medium throughput binding assay
• No optical interference from compounds
190, 191
  Fluorescence polarization • Assay reported in literature using specific commercial reagents
• Substrate: fluorescent BODIPY labelled tracer
• Detection: BRD4/BODIPY labelled tracer complex
• Medium sensitivity
• Medium throughput binding assay
190
  TR-FRET • Commercial validated assay kit
• Substrate: biotinylated peptide
• Detection: BRD4/biotinylated peptide complex
• High sensitivity
• High throughput binding assay
192