TCH_009814_01_LANCE_46_Final

T e c h n i c a l
N o t e
U-TRF #46
LANCE Ultra HDAC2 Histone H3 Lysine
4 Deacetylase Assay
Authors
Julie Blouin
Mireille Caron
Lucille Beaudet
PerkinElmer, Inc.
Montreal, QC
Canada, H3J 1R4
LANCE® Ultra
This LANCE Ultra immunodetection assay measures the deacetylation
of a biotinylated Histone H3 (1-21) peptide acetylated at lysine 4.
Ac
K
B
Europium-anti-unmodified Histone H3 Lysine 4 (H3K4)
Antibody
+ Enzyme
• TRF0404-D: 10 µg, 1,562 assay points*
• TRF0404-M: 100 µg, 15,625 assay points*
*40 fmol/assay point
K
B
Peptidic Substrate Sequence:
+ Eu-Ab
+ ULight-SA
ARTK(ac)QTARKSTGGKAPRKQLA-GG-K(BIOTIN)-OH
LANCE Ultra Assays
LANCE Ultra time-resolved fluorescence resonance energy transfer
(TR-FRET) assays use a proprietary europium chelate donor dye,
W1024 (Eu), together with ULight™, a small molecular weight
acceptor dye with a red-shifted fluorescent emission.
In this technical note, we present the optimization of an epigenetic
enzymatic assay using a biotinylated Histone H3-derived peptide
as substrate. The deacetylated peptide product is captured by the
Eu-labeled antibody (Eu-Ab) and ULight-Streptavidin (ULight-SA)
which bring the Eu donor and ULight acceptor dye molecules into
close proximity. Upon irradiation at 320 or 340 nm, the energy from
the Eu donor is transferred to the ULight acceptor dye which, in turn,
generates light at 665 nm. The intensity of the light emission is
proportional to the level of biotinylated substrate modification.
Eu
FRET
Emission
665 nm
ULight
B
Excitation
320 or 340 nm
K
Figure 1. Schematic representation of the LANCE Ultra detection of a
deacetylated histone peptide.
Development of a HDAC2 Histone H3-Lysine 4
Deacetylase Assay:
Reagents needed for the assay:
Europium-anti-unmodified Histone H3
Lysine 4 (H3K4) Antibody
PerkinElmer # TRF0404
LANCE Ultra ULight-Streptavidin
PerkinElmer # TRF0102
Histone H3 (1-21), H3K4ac
peptide, biotinylated
AnaSpec # 65207
LANCE Detection Buffer, 10X
PerkinElmer # CR97-100
HDAC2 (human), recombinant
BPS BioScience # 50002
Trichostatin A
Sigma # T-8552
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 DDT, 0.01%
Tween-20 and 0.01% BSA.
Standard Protocol
• Dilute HDAC2 enzyme, inhibitors and biotinylated Histone H3K4ac 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 room temperature (RT).
– 5 μL of biotinylated Histone H3K4ac peptide (2X)
• Cover the plate with TopSeal-A film and incubate at RT
• Prepare a 4X Stop Solution containing 12 µM of Trichostatin A in 1X LANCE
Detection Buffer (final concentration of 3 µM Trichostatin A in
20 µL total assay volume).
• Prepare a 4X Detection Mix by diluting the Eu-Ab to 8 nM and ULightStreptavidin to 200 nM in 1X LANCE Detection Buffer (final concentrations
of 2 nM and 50 nM, respectively, in 20 µL total assay volume). Add to the wells:
– 5 μL of Trichostatin A Stop Solution. Incubate 5 min at RT
– 5 μL of Detection Mix
• Cover with TopSeal-A film and incubate for 60 min at RT.
• Remove the TopSeal-A film and read signal with the EnVision®
Multilabel Reader in TR-FRET mode (excitation at 320 or 340 nm
& emission at 665 nm).
Experiment 1: Enzyme Titration and Time-Course
Experiment 2: Enzyme Inhibition
Enzymatic progress curves were performed by incubating HDAC2 at concentrations ranging from 2.5 to 20 nM with 1 µM biotinylated Histone H3K4ac peptide
substrate. Reactions were stopped by the addition of Trichostatin A at indicated
times. Detection mix was then added and signal read after 60 min. A 60 min
reaction time using 5 nM enzyme was selected for all subsequent experiments.
Serial dilutions of Trichostatin A ranging from 10 pM to 100 µM and serial dilutions
of SAHA ranging from 100 pM to 1 mM were pre-incubated for 5 min with 5 nM of
HDAC2. Enzymatic reactions were initiated by the addition of 1 µM biotinylated
Histone H3K4ac peptide substrate. Enzymatic reactions contain 2% DMSO.
Experiment 3: Z'-factor Determination
HDAC2 (5 nM) was pre-incubated with or without 3 µM Trichostatin A for
5 min. Enzymatic reactions were initiated by the addition of 1 µM biotinylated
Histone H3K4ac peptide substrate. Enzymatic reactions contain 2% DMSO.
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009814_01
Printed in USA
Aug. 2011