AlphaLISA SIRT1 p53 Lysine 382vDeacetylase Assay

T E C H N I C A L
N O T E
AlphaLISA #10
AlphaLISA SIRT1 p53 Lysine 382
Deacetylase Assay
Authors
Mathieu Arcand
Julie Blouin
Mireille Caron
Claire Normand
Anne Labonté
Lucille Beaudet
Jaime Padrós
AlphaLISA®
PerkinElmer, Inc.
Montreal, QC
Canada, H3J 1R4
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 SIRT1 assay using as substrate a
biotinylated p53-derived peptide acetylated at lysine 382. In the
absence of enzyme or cofactor, the anti-p53K382ac 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 enzyme and cofactor are added to the reaction, the
peptide substrate is deacetylated and the anti-p53K382ac 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 SIRT1 enzyme.
This AlphaLISA immunodetection assay measures the deacetylation
of a biotinylated p53 (368-393) peptide acetylated at lysine 382.
Anti-acetyl-p53 Lysine 382 (p53K382ac) AlphaLISA®
Acceptor Beads
• AL124C: 250 µg, 500 assay points*
• AL124M: 5 mg, 10,000 assay points*
• AL124R: 25 mg, 50,000 assay points*
*0.5 µg/assay point
Peptidic Substrate Sequence:
(Biotin)K-GG-HLKSKKGQSTSRHKK(ac)LMFKTEGPDSD-NH2
Ac
K
B
No
Enzyme
+ Enzyme
NAD+
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 p53-derived peptide.
K
Development of a SIRT1 p53 Lysine 382 Deacetylase Assay:
Reagents needed for the assay:
Anti-acetyl-p53 Lysine 382
AlphaLISA Acceptor beads
PerkinElmer # AL124
p53 (368-393) acetyl-lysine 382
peptide (p53K382ac), biotinylated
AnaSpec # 64869
Alpha Streptavidin Donor beads
PerkinElmer # 6760002
AlphaLISA 5X Epigenetics Buffer 1 Kit
PerkinElmer # AL008
Sirtuin 1 (human), recombinant
BPS BioScience # 50012
EX-527
Tocris # 2780
Suramin
Calbiochem # 574625
SIRT1 inhibitor III
Calbiochem # 566322
Nicotinamide
Sigma # N3376
ß-Nicotinamide adenine dinucleotide
hydrate (NAD+)
Sigma # N1636
White opaque OptiPlate™-384
PerkinElmer # 6007299
TopSeal™-A films
PerkinElmer # 6005185
Assay Buffer: 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM DTT, 0.01%
Tween-20 and 0.01% BSA.
250,000
Experiment 3: Enzyme Inhibition
[SIRT1] (nM)
200,000
0
0.25
0.5
1
2
5
150,000
100,000
50,000
0
0
20
40
60
80
Time (min)
100
120
AlphaLISA Signal (counts)
AlphaLISA Signal (counts)
Experiment 1: Enzyme Titration and Time-Course
Standard Protocol
• Dilute SIRT1 enzyme, inhibitors, biotinylated p53K382ac peptide substrate
and NAD+ 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
– 2.5 μL of biotinylated p53K382ac peptide substrate (4X)
– 2.5 µL of NAD+ (4X)
• 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 Stop Solution Mix containing 250 µM of EX-527 and 100 μg/mL
of Acceptor Beads in 1X Epigenetics Buffer 1 (final concentration of 50 µM
EX-527 and 20 μg/mL Acceptor Beads in 25 μL total assay volume).
– 5 μL of Stop Solution Mix
• 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.
200,000
EX-527
IC50 = 1.09 µM
150,000
SIRT1 Inhibitor III
IC50 = 0.81 µM
100,000
Suramin
IC50 = 0.13 µM
50,000
0
Nicotinamide
IC50 = 91 µM
-∞
-9
-8
-7
-6
-5
Log [Inhibitor] (M)
-4
-3
-2
Experiment 2: NAD+ Titration
Experiment 4: Z’-factor Determination
250,000
200,000
150,000
100,000
Km app = 167 µM
50,000
0
-∞ -7
-6
-5
-4
Log [NAD+] (M)
-3
-2
Serial dilutions of NAD+ ranging from 300 nM to 12.5 mM were added to 0.5 nM
SIRT1 and 3 nM biotinylated p53K382ac peptide substrate. Enzymatic reactions
were incubated for 30 min. A 200 µM NAD+ concentration was selected for
subsequent experiments.
AlphaLISA Signal (counts)
Serial dilutions of inhibitors ranging from 1 nM to 100 µM (EX-527), 3 nM to 100 µM
(SIRT1 Inhibitor III), 10 nM to 10 µM (suramin) and 300 nM to 3 mM (nicotinamide) were pre-incubated for 5 min with 0.5 nM of SIRT1. Enzymatic reactions were
initiated by the addition of 3 nM biotinylated p53K382ac peptide substrate and 200
µM NAD+. Enzymatic reactions contained 1% DMSO and proceeded for 60 min.
AlphaLISA Signal (counts)
Enzymatic progress curves were performed by incubating SIRT1 at concentrations ranging from 0.25 to 5 nM with 3 nM biotinylated p53K382ac peptide
substrate and 2 mM NAD+. A mix of Acceptor beads and EX-527 was added to
stop the reaction at the indicated times. Streptavidin Donor beads were added 60
min later and signal was read after 30 min. A 0.5 nM enzyme was selected for all
subsequent experiments.
200,000
30 µM EX-527
Z' = 0.55
SB = 2.6
150,000
100,000
50,000
0
No inhibitor
0
10
20
For a complete listing of our global offices, visit www.perkinelmer.com/ContactUs
Copyright ©2010-2011, PerkinElmer, Inc. All rights reserved. PerkinElmer® is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners.
Printed in USA
Apr. 2011
40
50
SIRT1 (0.5 nM) was pre-incubated with or without 30 µM EX-527 for 5 min.
Enzymatic reactions were initiated by the addition of 3 nM biotinylated p53K382ac
peptide substrate and 200 µM NAD+. Enzymatic reactions contained 1% DMSO and
proceeded for 60 min.
PerkinElmer, Inc.
940 Winter Street
Waltham, MA 02451 USA
P: (800) 762-4000 or
(+1) 203-925-4602
www.perkinelmer.com
009615_01
30
Well #
Similar pages