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