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 |