(PerkinElmer) technologies (where Alpha stands for Amplified Luminescent Proximity Homogeneous Assay Screen). These are non-enzymatic in vitro assays that
detect protein interactions using donor and acceptor beads. When the interaction
takes place, a chemical reaction produces an amplified signal. After donor excitation,
singlet state oxygen molecules react with acceptor fluorophores, if located in proximity, that emit light at 520–620 nm [8]. Generally, donor beads in AlphaScreen and
AlphaLISA contain a phthalocyanine molecule that produces a singlet state oxygen,
keeping energy irradiation at 680 nm. Acceptor beads contain thioxene, anthracene
and rubene in AlphaScreen and thioxene, europium, terbium or samarium in
AlphaLISA [9]. The beads are covered with reactive aldehydes to bind the ligand,
antibody or substrate [9]. Compared to other methods such as time-resolved fluorescent energy transfer (TR-FRET), these assays have better sensitivity and can be
used for large-scale reactions. Depending on the interaction under investigation, the
beads are functionalized in different ways. AlphaLISA epigenetic toolbox
(PerkinElmer) is used to detect histone methylation and acetylation marks. Using
S-adenosylmethionine or acetyl CoA as a cofactor, epigenetic enzymes such as
histone methyltransferases (HMTs) or histone acetyltransferases (HATs) react on
the specific biotinylated histone substrate. Antibody AlphaLISA acceptor beads and
streptavidin Alpha donor beads are then added to measure methylation and acetylation levels. The donor binds the histone substrate by biotin-streptavidin binding and
the acceptor captures the modification. If the enzyme works, the acceptor and donor
beads are in proximity, and, after laser irradiation, a chemiluminescent signal at
615 nm is generated [10]. The same methodology was used to develop an HTS
platform to identify new G9a [9] and JMJD2A [11] modulator drugs.
AlphaScreen and AlphaLISA offer several advantages:
• The difference between donor and acceptor wavelength reduces interference.
• Interactions are detected from sub-nM to μM range.
• The assay is suitable for use in HTS and adapts to 96-, 384- and 1,536-well
formats.
• Many biological interactions can be studied.
1.1.2 Surface Plasmon Resonance (SPR)
To study in real time the association and dissociation constants of protein complexes
or drug enzymes, SPR is one of the most widely used in vitro assays. The technique
measures refractive index changes when there is a variation in mass on a gold chip.
Incident light (Fig. 1) strikes electrons at the surface of the metal sensor and converts
them into surface plasmon waves, generating an SPR angle. Interaction between the
target immobilized on the chip and the captured analyte induces a perturbation at the
gold surface and therefore a change in angle of reflection, which can be measured.
To improve the optical quantitative detection method, SPR microscopy or imaging
(SPRi) technology is used for high-throughput probing of biomolecular interactions.
SPRi Biochips™ and SPRi Slides™ are customized for HORIBA SPR imaging
Advanced Assays in Epigenetics
527
detect protein interactions using donor and acceptor beads. When the interaction
takes place, a chemical reaction produces an amplified signal. After donor excitation,
singlet state oxygen molecules react with acceptor fluorophores, if located in proximity, that emit light at 520–620 nm [8]. Generally, donor beads in AlphaScreen and
AlphaLISA contain a phthalocyanine molecule that produces a singlet state oxygen,
keeping energy irradiation at 680 nm. Acceptor beads contain thioxene, anthracene
and rubene in AlphaScreen and thioxene, europium, terbium or samarium in
AlphaLISA [9]. The beads are covered with reactive aldehydes to bind the ligand,
antibody or substrate [9]. Compared to other methods such as time-resolved fluorescent energy transfer (TR-FRET), these assays have better sensitivity and can be
used for large-scale reactions. Depending on the interaction under investigation, the
beads are functionalized in different ways. AlphaLISA epigenetic toolbox
(PerkinElmer) is used to detect histone methylation and acetylation marks. Using
S-adenosylmethionine or acetyl CoA as a cofactor, epigenetic enzymes such as
histone methyltransferases (HMTs) or histone acetyltransferases (HATs) react on
the specific biotinylated histone substrate. Antibody AlphaLISA acceptor beads and
streptavidin Alpha donor beads are then added to measure methylation and acetylation levels. The donor binds the histone substrate by biotin-streptavidin binding and
the acceptor captures the modification. If the enzyme works, the acceptor and donor
beads are in proximity, and, after laser irradiation, a chemiluminescent signal at
615 nm is generated [10]. The same methodology was used to develop an HTS
platform to identify new G9a [9] and JMJD2A [11] modulator drugs.
AlphaScreen and AlphaLISA offer several advantages:
• The difference between donor and acceptor wavelength reduces interference.
• Interactions are detected from sub-nM to μM range.
• The assay is suitable for use in HTS and adapts to 96-, 384- and 1,536-well
formats.
• Many biological interactions can be studied.
1.1.2 Surface Plasmon Resonance (SPR)
To study in real time the association and dissociation constants of protein complexes
or drug enzymes, SPR is one of the most widely used in vitro assays. The technique
measures refractive index changes when there is a variation in mass on a gold chip.
Incident light (Fig. 1) strikes electrons at the surface of the metal sensor and converts
them into surface plasmon waves, generating an SPR angle. Interaction between the
target immobilized on the chip and the captured analyte induces a perturbation at the
gold surface and therefore a change in angle of reflection, which can be measured.
To improve the optical quantitative detection method, SPR microscopy or imaging
(SPRi) technology is used for high-throughput probing of biomolecular interactions.
SPRi Biochips™ and SPRi Slides™ are customized for HORIBA SPR imaging
Advanced Assays in Epigenetics
527