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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-Gloâ„¢ 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 (LanthaScreenâ„¢)

• 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 (Epigenaseâ„¢)

• 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 (AlphaScreenâ„¢)

• 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 (BromoMELTâ„¢)

• 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