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HTS Assays
for Epigenetic Enzymes
Assays used for Epigenetic Enzymes
Transcreener® & Epigenetic Modifications
3
Transcreener relies on direct detection
of ADP. Binding of tracer to antibody
causes a change in fluorescence. There
are just two components, and no
intermediate steps.
Mix-and-Read protocol
ADP2 Assays:
Direct detection of ADP with FP, TR-FRET or FI Readouts
0.01 0.1 1 100
50
100
150
200
250
0 hr
1hr
4hr
8hr
24hr
ADP (µM)
m
P
0.01 0.1 1 100
50
100
150
200
250
RT
37°C
Control
-80°C
-20°C
4°C
ADP (µM)
m
P
21 Day Reagent Stability
Signal Stability
% ATP Conversion 0.1 µM 1 µM 10 µM 100 µM 1000 µM
100 0.96 0.95 0.95 0.94 0.93
60 0.94 0.94 0.95 0.94 0.93
30 0.89 0.94 0.94 0.92 0.9
20 0.87 0.94 0.93 0.92 0.9
15 0.87 0.93 0.92 0.91 0.89
10 0.78 0.91 0.93 0.88 0.89
7.5 0.74 0.87 0.92 0.89 0.85
5 0.65 0.86 0.88 0.87 0.83
3 0.53 0.76 0.85 0.82 0.8
1.5 -4.22 0.36 0.6 0.63 0.62
0.75 -2.24 0.25 0.48 0.54 0.6
ADP2 Assay: Stable, Robust and Reproducible
Z` Values
0 50 100 150 200 2500
50
100
150
200
Kemptide-50 M
Histone H1-3 M
Histone H3-10M
[PKA] (ng/mL)
m
P
Histone phosphorylation can be interrogated
using the Transcreener ADP2 assay.
Exploring Histone Modification (phosphorylation)
From Tang Z., et al, 2008. FEBS Journal 275: 3003-3015.
AMP2/GMP2 Assay: Monitoring AMP Flux
Transcreener Assay:
In this study: SUMO= SUMO-1 E1= SAE1/SAE2 E2 = Ubc9 E3= RanBP2
Sumoylation, Neddylation, and Ubiquitination Pathways
AMP2/GMP2 Assay:
Eq.1 E1 + ATP + Sumo E1.AMP~Sumo + PPi
Eq.2 E1.AMP~Sumo E1-S-CO-Sumo + AMP
Eq.3 E1-S-CO-Sumo + ATP + Sumo E1-S-CO-Sumo + PPi.AMP-Sumo
0 50 100 150 200 250 3000
50
100
150
200
250
0
100
200
300
400
+ Sumo
- Sumo
E1, nM
m
P
AM
P, n
M
Reactions were allowed to go to completion, thus AMP should be produced
in stoichiometric amounts with E1, as was observed.
E1, nM
ExperimentalAMP, nM
TheoreticalAMP, nM
150 134 (±8.6) 150
75 69 (±4.2) 75
37.5 41 (±7.4) 37.5
18.7 19 (±4.1) 18.7
9.3 8 (±3.1) 9.3
An increase in the polarization signal
(ΔmP) is observed with increasing E1
in the presence of SUMO.
AMP Formation is SUMO-dependent and stoichiometric with E1
AMP2/GMP2 Assay:
E1-S-CO-Sumo
.AMP-Sumo .AMP-Sumo
Eq.5 + RanBP2-NH2 E2-SH + RanBP2-NH-CO-Sumo
A. B.
Reactions were again run to completion. Addition of E2 resulted in a stoichiometric increase in
AMP, as expected (Eq. 4). Addition of E3 to the reaction resulted in >5-fold increase in AMP
production, suggesting that multiple SUMOs are being transferred to E3.
E2, nM
Experimental+ 50 nM E1AMP, nM
Theoretical*+ 50 nM E1AMP, nM
Experimental+ 50 nM E1 + 75 nM E3
AMP, nM
Theoretical*+50 nM E1+ 75 nM E3
AMP, nM
50 119 (±7.2) 100 758 (±4.8) 175
25 86 (±4.3) 75 600 (±6.2) 150
12.5 61 (±5.4) 62.5 368 (±7.1) 137.5
0 47 (±3.4) 50 53 (±1.4) 50
AMP Production by E1 is enhanced by E2 and E3
Leverages the HTS proven Transcreener AMP/GMP Assay for universal
detection of methyltransferases in a mix-and-read format.
www.bellbrooklabs.com
www.reactionbiology.com
: Turn key assays combining
Reaction Biology’s purified enzymes and BellBrook’s Transcreener EPIGEN Assay
Precalibrated Enzyme
Optimal Substrate
Reaction Buffer, SAM, etc.
Transcreener EPIGEN Detection Reagents
Quick Guide with lot specific enzyme info
0.01 1 1000
50
100
150
200G9a/H3(1-21)
SET8/H4 (15-24)
Set7/9/H3(1-21)
NSD2/Nucleosomes
MLL4 Complex/H3(1-21)
EZH2 Complex/H3(21-44)
SUV39H1/H3(1-21)
Dot1L/nucleosomes
GLP/H3(1-21)
G9a/GLP/H3(1-21)
[E], ng/L
m
P
0.01 0.1 1 100
50
100
150
200
PRMT1/H4(1-20)
PRMT3/H4(1-20)
PRMT8/GST-GAR
PRMT4/H3-3
[E] ng/L
m
P
0.1 1 10 1000
50
100
150
DNMT1
DNMT3b
ng/L
m
P
HMT screening and profiling with no assay development
Enzyme, ng/L
SA
H,
M
0.0 0.5 1.0 1.5 2.0 2.50.0
0.1
0.2
0.3
0.4
0.5
G9a
SET8
SET7/9
PRMT3
Using the precalibrated EC85 concentration of enzyme will provide an outstanding assay window and insure initial velocity conditions, eliminating the need to use precious enzyme for time consuming assay development.
0 50 100 150 200 250 300 350
0
25
50
75
100
125
without acceptor substrate
1 M Histone H3 (full length)
10 M Histone H3 Peptide 1-25
100 M Histone H3 Peptide 1-25
10 M Histone H3 Peptide 15-39
[G9a] ng/mL
m
P
Acetyltransferases
Active Inactive
11
The EPIGEN Acetyltransferase Assay
Leverages the HTS proven Transcreener
AMP/GMP Assay for universal HAT
detection.
EPIGEN Acetyltransferase Assay:
0 10 20 30 40 50 600
25
50
75
100
2
1
0.5
0.25
0.13
0.060
[pCAF] ng/L
Time, min
m
P
10 µM peptide + 100 µM Acetyl CoA
0 10 20 30 40 50 600
2
4
6
8
10
12
0.5
0.25
0.125
0.0625
0
1
[pCAF] ng/L
Time, min
Co
A,
M
EPIGEN Acetyltransferase Assay:
The enzymatic progress curves were performed by incubating pCAF enzyme,
H3 peptide, and Acetyl CoA.
They were stopped by the addition of a detection mix containing an
AMP2/GMP2 antibody and tracer, the coupling enzyme system, and Anacardic
Acid at the indicated time points.
The Signal was read after 60 minutes. A 60 minute enzyme reaction with pCAF
was selected for all subsequent experiments. Controls lacked the H3 peptide.
Enzyme and Time Dependent Production of CoA
13
0 20 40 60 80 1000
50
100
150
200
0.5 Hr
1 Hr
2 Hr
3 Hr
24 Hr
CoA M
m
P
Signal Stability
0 2 4 6 8 100
50
100
150
200
0.0
0.2
0.4
0.6
0.8
1.0
CoA, M
m
P
Z' V
alu
e
0 20 40 60 80 1000
50
100
150
200
0.0
0.2
0.4
0.6
0.8
1.0
CoA, M
m
P
Z' V
alu
e
10 µM µAcetyl CoA/CoA Standard
Curve
100 µM Acetyl CoA/ CoA Standard Curve
Excellent Z’ Values
EPIGEN Acetyltransferase Assay:
Technology offers simple solutions for interrogating epigenetic mechanisms.
Universal: Any Enzyme, Any substrate, Any ATP/SAM concentration
Sensitive: low substrate consumption, use less enzyme
Direct detection, far red fluors: less interference
Single addition, mix and read format: easy automation
Three fluorescent readouts, instrument-validated: flexibility, confidence
> 12hr reagent and signal stability: easy automation
For more information on Transzyme Methyltransferase Assay Kits, email us at
[email protected] or call toll free 866-313-7881.
Visit our website at www.bellbrooklabs.com
