T E C H N I C A L N O T E
AlphaLISA KAT5 (TIP60) Assay
AlphaLISA® Technology
AlphaLISA #30
Mireille Caron Marie-Élaine Caruso Nancy Gauthier Anja Rodenbrock Philippe Bourgeois Liliana Pedro Lucille Beaudet Roberto Rodriguez-Suarez
PerkinElmer, Inc. Montreal, QC Canada H3J 1R4
This AlphaLISA immunodetection assay measures the acetylation of a Histone H4 (1-25) peptide at the N-terminal lysine residues.
Anti-Acetyl-Lysine AlphaLISA Acceptor Beads
• AL143C:250μg,500assaypoints*
• AL143M:5mg,10,000assaypoints*
• AL143R:25mg,50,000assaypoints*
*0.5μg/assaypoint
Peptidic Substrate Sequence:
SGRGKGGKGLGKGGAKRHRKVLRDNGSGS-K(Biotin)
AlphaLISA Assays
AlphaLISAtechnologyisapowerfulandversatileplatformthatoffershighlysensitive,no-washimmunoassaysusingAlphaDonorandAlphaLISAAcceptorbeads.Inthistechnicalnote,wepresenttheoptimizationofaKAT5(TIP60)enzymaticassayusingabiotinylatedH4-derivedpeptideassubstrate.DetectionoftheacetylatedproductwasperformedbytheadditionofStreptavidin(SA)AlphaDonorbeadsandAlphaLISAAcceptorbeadsconjugatedtoanantibody(Ab)directedagainstacetylatedlysineresidues.Uponlaserirradiationofthebeads-targetcomplexesat680nm, short-livedsingletoxygenmoleculesproducedbytheDonorbeadscanreach theAcceptorbeadsinproximitytogenerateanamplifiedchemiluminescent signalat615nm.TheintensityofthelightemissionisproportionaltotheacetylationactivityoftheKAT5(TIP60)enzyme.
+ Enzyme
+ SA-Donor Bead+ Ab-Acceptor Bead
AcCoA
CoA
K
K
K
Excitation680 nm
B
B
B
Emission615 nm
Ac
Ac
Figure 1. Schematic representation of the AlphaLISA detection of an acetylated histone peptide (B: biotin group; K: lysine residue).
Acceptor
Donor
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Development of AlphaLISA KAT5 (TIP60) AssayReagents needed for this assay: AlphaLISAanti-Acetyl-LysineAcceptorbeads PerkinElmer#AL143
AlphaStreptavidinDonorbeads PerkinElmer#6760002
HistoneH4(1-25)peptide,biotinylated AnaSpec#65242
AlphaLISA5XEpigeneticsBuffer1Kit PerkinElmer#AL008
KAT5(TIP60),active SignalChem#K314-380G
WhiteopaqueOptiPlate™-384 PerkinElmer#6007290
TopSeal™-Afilm PerkinElmer#6050195
AcetylcoenzymeAsodiumsalt Sigma#A2056
Anacardicacid EMDMIllipore#172050
Garcinol Sigma#G5173
AssayBuffer:50mMTRIS-HClpH8.0,0.1mMEDTA,1mMDTT,330nMTSA,0.01%BSAand0.01%Tween
Standard Protocol• DiluteKAT5(TIP60)enzyme,acetylCoA,inhibitorsandbiotinylatedH4(1-25)substrateinAssayBufferjustbeforeuse.
• AddtothewellsofawhiteOptiPlate-384: –2.5μLofinhibitor(4X)orAssayBuffer –5μLofenzyme(2X) –Incubatefor5minat23°C. –2.5μLofbiotinylatedH4(1-25)/acetylCoAmix(4X)
For acetyl CoA titration, add acetyl CoA dilutions independently of substrate.
• CovertheplatewithTopSeal-Afilmandincubateat23°C.• Prepare1XEpigeneticsBuffer1asrecommendedinthebuffer
technical data sheet.• PrepareAcceptorbeadsat100µg/mLin1XEpigeneticsBuffer1(finalconcentrationof20µg/mLin25µLtotalassayvolume).
• Add5μLofAcceptorbeads.Addition of Acceptor beads prepared in Epigenetics Buffer 1 stops the enzymatic reaction.
• CoverwithTopSeal-Afilmandincubate60minat23°C.• PrepareStreptavidinDonorbeadsat50µg/mLin1XEpigeneticsBuffer1insubduedlight(finalconcentrationof20µg/mLin25µLtotalassayvolume).
• Add10µLofDonorbeadsinsubduedlight.• CoverwithTopSeal-Afilmandincubateinsubduedlightfor30minat23°C.• ReadsignalinAlphamodewiththeEnVision®MultilabelPlateReaderorEnSpire®MultimodePlateReader.
Experiment 1: Enzyme Titration and Time Course
Enzymatic progress curves were performed by incubating KAT (TIP60) at concentrations ranging from 0.1 to 10 nM with 100 nM biotinylated H4 (1-25) substrate. Acceptor beads were added at the indicated times. Donor beads were added 60 min later and signal was read after 30 min. A 60 min reaction time using 1 nM enzyme was selected for all subsequent experiments.
Experiment 2: Acetyl CoA Titration
Serial dilutions of acetyl CoA ranging from 1 nM to 30 µM were added to 1 nM KAT5 (TIP60) and 100 nM biotinylated H4 (1-25) peptide substrate. A 1.5 µM acetyl CoA concentration was selected for subsequent experiments.
Experiment 4: Z’-factor Determination
KAT5 (TIP60) (1 nM) was pre-incubated with or without 100 µM anacardic acid for 5 min. Enzymatic reactions were initiated by the addition of 100 nM biotinylated H4 (1-25) peptide substrate. Enzymatic reactions contain 1% DMSO.
Experiment 3: Enzyme Inhibition
Serial dilutions of anacardic acid and garcinol ranging from 10 nM to 1 mM and 3 nM to 0.3 mM, respectively, were pre-incubated for 5 min with 1 nM KAT5 (TIP60). Enzymatic reactions were initiated by the addition of 100 nM biotinylated H4 (1-25) peptide substrate. Enzymatic reactions contain 1% DMSO.
Results