Lance Ultra SET7/9 Histone H3-Lysine N

T e c h n i c a l
N o t e
U-TRF #35
LANCE Ultra SET7/9 Histone H3-Lysine
N-methyltransferase Assay
Authors
Nancy Gauthier
Valérie Paquet
Liliana Pedro
Anne Labonté
Anja Rodenbrock
Marjolaine Roy
Lucille Beaudet
Roberto Rodriguez-Suarez
LANCE® Ultra
PerkinElmer, Inc.
Montreal, QC
Canada, H3J 1R4
This LANCE® Ultra immunodetection assay measures the
mono-methylation of a biotinylated Histone H3 (1-21) peptide
at lysine 4.
K
B
+ Enzyme
Europium-anti-methyl-Histone H3 Lysine 4 (H3K4me1-2)
Antibody
• TRF0402-D: 10 µg, 1,562 assay points*
• TRF0402-M: 100 µg, 15,625 assay points*
*40 fmol/assay point
SAM
SAH
Me
K
B
Peptidic Substrate Sequence:
ARTKQTARKSTGGKAPRKQLA-GG-K(BIOTIN)-NH2 + Eu-Ab
+ ULight-SA
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.
Eu
In this technical note, we present the optimization of an epigenetic
enzymatic assay using a biotinylated histone H3-derived peptide as
substrate. The modified peptide is captured by the Eu-labeled antibody
(Eu-Ab) and ULight-Streptavidin (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.
FRET
Em 665 nm
Excitation
320 or 340 nm
Me
ULight
B
K
Figure 1. Schematic representation of the LANCE Ultra detection of a modified
histone peptide.
Development of a SET7/9 Histone H3-Lysine
N-methyltransferase Assay
Reagents needed for the assay:
Europium-anti-methyl-Histone H3
Lysine 4 (H3K4me1-2)
LANCE Ultra ULight-Streptavidin
Histone H3 (1-21) peptide, biotinylated
LANCE Detection Buffer, 10X
SET7/9 (human), recombinant
White opaque OptiPlate™-384
TopSeal™-A films
S-(5'-Adenosyl)-L-methionine chloride (SAM)
Sinefungin
S-(5'-Adenosyl)-L-homocysteine (SAH)
PerkinElmer # TRF0402
PerkinElmer # TRF0102
AnaSpec # 61702
PerkinElmer # CR97-100
Enzo # ALX-201-178-C100
PerkinElmer # 6007299
PerkinElmer # 6005185
Sigma # A7007
Sigma # S8559
Sigma # A9384
Standard Protocol
• Dilute SET7/9 enzyme, SAM, inhibitors and biotinylated peptide
substrate in Assay Buffer just before use.
• Add to the wells of a white Optiplate-384:
– 5 μL of inhibitor (2X) or Assay Buffer
– 2.5 μL of enzyme (4X)
– 2.5 μL of biotinylated Histone H3 (1-21) peptide/SAM mix (4X).
For SAM titration, add SAM dilutions independently of substrate.
• Cover the plate with TopSeal-A film and incubate at room temperature (RT).
• Prepare Detection Mix* by diluting the Eu-Ab to 4 nM and ULight-Streptavidin
to 100 nM in 1X LANCE Detection Buffer (final concentrations of 2 nM and
50 nM respectively, in 20 µL total assay volume).
• Add 10 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 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 3: Enzyme Inhibition
70,000
[SET7/9] (nM)
60,000
10
5
2.5
1
0.5
0
50,000
40,000
30,000
20,000
10,000
0
LANCE Signal (665 nm)
* Under these experimental conditions, addition of the Detection Mix stops
SET7/9 enzymatic reaction.
LANCE Signal (665 nm)
SAM is prepared at 30 mM in 5 mM H2SO4/10% ethanol (v/v) in H2O,
aliquoted and stored at -80 °C.
Assay Buffer: 50 mM Tris-HCl, pH 8.8, 5 mM MgCl2, 1 mM DTT, 0.01%
Tween-20
60,000
Sinefungin
IC50 = 769 nM
50,000
40,000
SAH
IC50 = 93 μM
30,000
20,000
10,000
0 10 20 30 40 50 60 70 80 90 100
0
-∞
-9
-8
-7
-6
-5
-4
-3
Log [Inhibitor] (M)
Time (min)
Experiment 2: SAM Titration
Experiment 4: Z’-factor Determination
70,000
60,000
LANCE Signal (665 nm)
Serial dilutions of sinefungin ranging from 1 nM to 1 mM and SAH ranging from
10 nM to 1 mM were pre-incubated for 10 min with 5 nM SET7/9. Enzymatic
reactions were initiated by the addition of 200 nM biotinylated H3 (1-21) peptide
substrate plus 300 nM SAM. Enzymatic reactions contain 1% DMSO.
LANCE Signal (665 nm)
Enzymatic progress curves were performed by incubating SET7/9 at concentrations
ranging from 0.5 to 10 nM with 200 nM biotinylated H3 (1-21) peptide substrate
and 300 μM SAM. Detection Mix was added to stop the reactions at the indicated
times and signal was read after 60 min. A 60 min reaction time using 5 nM enzyme
was selected for all subsequent experiments.
Km app = 75 nM
50,000
40,000
30,000
20,000
10,000
0
-∞ -11 -10 -9
-8
-7
-6
-5
-4
Log [SAM] (M)
Serial dilutions of SAM ranging from 30 pM to 100 µM were added to 5 nM
SET7/9 and 200 nM biotinylated H3 (1-21) peptide substrate. A 300 nM SAM
concentration was selected for subsequent experiments.
60,000
No inhibitor
50,000
40,000
30,000
20,000
300 µM Sinefungin
10,000
0
0
10
20
Copyright © 2010, PerkinElmer, Inc. All rights reserved. PerkinElmer® is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners.
Oct. 2010
40
50
SET7/9 (5 nM) was pre-incubated with or without 300 µM sinefungin for 10 min.
Enzymatic reactions were initiated by the addition of 200 nM biotinylated H3 (1-21)
peptide substrate plus 300 nM SAM. Enzymatic reactions contain 1% DMSO.
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009371_01
Z' = 0.88
S/B = 9.1