AlphaLISA HDAC1 Histone H3-Lysine 27 Deacetylase Assay

T E C H N I C A L
N O T E
AlphaLISA #6
AlphaLISA HDAC1 Histone H3-Lysine 27
Deacetylase Assay
Authors
Julie Blouin
Mathieu Arcand
Mireille Caron
Anne Labonté
Claire Normand
Lucille Beaudet
Jaime Padrós
AlphaLISA®
PerkinElmer, Inc.
Montreal, QC
Canada, H3J 1R4
This AlphaLISA immunodetection assay measures the deacetylation of
a biotinylated Histone H3 (21-44) peptide acetylated at lysine 27.
Anti-acetyl Histone H3 Lysine 27 (H3K27ac) AlphaLISA®
Acceptor Beads
• AL120C: 250 µg, 500 assay points*
• AL120M: 5 mg, 10,000 assay points*
• AL120R: 25 mg, 50,000 assay points*
*0.5 µg/assay point
Peptidic Substrate Sequence:
ATKAARK(ac)SAPATGGVKKPHRYRP-GG-K(Biotin)-OH
AlphaLISA Assays
AlphaLISA technology is a powerful and versatile platform that offers
highly sensitive, no-wash immunoassays using Alpha Streptavidin Donor
and AlphaLISA Acceptor beads. In this technical note, we present the
optimization of a signal decrease HDAC1 assay using as substrate a
biotinylated Histone H3-derived peptide acetylated at lysine 27. In
the absence of enzyme, the anti-H3K27ac Acceptor beads bind the
acetylated residue on the peptide. Upon laser irradiation of the
beads-target complexes at 680 nm, short-lived singlet oxygen molecules
produced by the Donor beads can reach the Acceptor beads in proximity
to generate maximal AlphaLISA signal at 615 nm (left panel). When
the enzyme is added to the reaction, the peptide substrate is
deacetylated and the anti-H3K27ac Acceptor beads do not recognize
the biotinylated peptide anymore, leading to a signal decrease
(right panel). This signal decrease is proportional to the deacetylation
activity of the HDAC1 enzyme.
Ac
K
B
No
Enzyme
+ Enzyme
Ac
K
B
K
B
+ SA-Donor Bead
+ Ab-Acceptor Bead
+ SA-Donor Bead
+ Ab-Acceptor Bead
No
Emission
Emission
615 nm
Excitation
680 nm
Excitation
680 nm
Ac
B
K
B
Figure 1. Schematic representation of the AlphaLISA detection of a modified histone peptide.
K
Development of a HDAC1 Histone H3-Lysine 27
Deacetylase Assay
Reagents needed for the assay:
Anti-acetyl Histone H3 lysine 27 (H3K27ac)
AlphaLISA Acceptor beads
PerkinElmer # AL120
Histone H3 (21-44), H3K27ac
peptide, biotinylated
AnaSpec # 64846
Alpha Streptavidin Donor beads
PerkinElmer # 6760002
AlphaLISA 5X Epigenetics Buffer 1 Kit
PerkinElmer # AL008
HDAC1 (human), recombinant
Cayman Chemical # 10009231
Trichostatin A
Sigma # T8552
SAHA
Cayman Chemical # 10009929
White opaque OptiPlate™-384
PerkinElmer # 6007299
TopSeal™-A films
PerkinElmer # 6005185
Assay Buffer: 50 mM Tris-HCl pH 8.0, 0.1 mM EDTA, 1 mM DTT, 0.01%
Tween-20 and 0.01% BSA.
1.2 x 1006
Experiment 2: Enzyme Inhibition
[HDAC1] (nM)
1,000,000
0
0.125
0.25
0.5
1
2
800,000
600,000
400,000
200,000
0
0
20
40
60
80
100
Time (min)
120
AlphaLISA Signal (counts)
AlphaLISA Signal (counts)
Experiment 1: Enzyme Titration and Time-Course
Standard Protocol
• Dilute HDAC1 enzyme, inhibitors and biotinylated Histone H3K27ac peptide
substrate in Assay Buffer just before use.
• Add to the wells of a white OptiPlate-384:
– 2.5 μL of enzyme (4X)
– 2.5 μL of inhibitor (4X) or Assay buffer
– Incubate 5 min at RT
– 5 μL of biotinylated Histone H3K27ac peptide substrate (2X)
• Cover the plate with TopSeal-A film and incubate at room temperature (RT).
• Prepare 1X Epigenetics Buffer 1 as recommended in the buffer technical
data sheet.
• Prepare a 5X Acceptor beads solution at 100 μg/mL in 1X Epigenetics
Buffer 1 (final concentration of 20 μg/mL in 25 μL total assay volume).
– 5 μL of Acceptor beads
Addition of Acceptor beads prepared in 1X Epigenetics Buffer 1 stops
the enzymatic reaction.
• Cover with TopSeal-A film and incubate for 60 min at RT.
• Prepare a 2.5X Streptavidin Donor beads solution at 50 μg/mL in 1X
Epigenetics Buffer 1 (final concentration of 20 μg/mL in 25 μL total assay
volume) in subdued light.
– 10 μL of Streptavidin Donor beads
• Cover with TopSeal-A film and incubate in subdued light for 30 min at RT.
• Read signal in Alpha mode with the EnVision® or EnSpire® reader.
1,000,000
800,000
Trichostatin A
IC50 = 3.2 nM
600,000
SAHA
IC50 = 161 nM
400,000
200,000
0
-∞ -12 -11 -10 -9 -8 -7 -6 -5 -4 -3
Log [Inhibitors] (M)
Enzymatic progress curves were performed by incubating HDAC1 at concentrations
ranging from 0.125 to 2 nM with 3 nM biotinylated Histone H3K27ac peptide
substrate. Acceptor beads were added at the indicated times. Donor beads were
added 60 min later and signal was read after 30 min. A 30 min reaction time using
1 nM enzyme was selected for all subsequent experiments.
Serial dilutions of Trichostatin A ranging from 1 pM to 10 µM and serial dilutions
of SAHA ranging from 10 pM to 100 µM were pre-incubated for 5 min with 1 nM
of HDAC1. Enzymatic reactions were initiated by the addition of 3 nM
biotinylated Histone H3K27ac peptide substrate. Enzymatic reactions contain 1%
DMSO.
AlphaLISA Signal (counts)
Experiment 3: Z’-factor Determination
1.2 x 1006
1,000,000
100 nM Trichostatin A
800,000
600,000
Z' = 0.69
S/B = 2.9
No inhibitor
400,000
200,000
0
0
10
20
30
Well #
40
50
HDAC1 (1 nM) was pre-incubated with or without 100 nM Trichostatin A for
5 min. Enzymatic reactions were initiated by the addition of 3 nM biotinylated
Histone H3K27ac peptide substrate. Enzymatic reactions contain 1% DMSO.
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009614_01
Printed in USA
Apr. 2011
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