High Throughput Homogenous Bioluminescent Assays For Monitoring The Concentrations of
AMP, ADP and ATP
1,2
Goueli ,
1
Hsiao ,
1
Zegzouti
Said A.
Kevin
and Hicham
Cell Signaling, Research and Development, Promega Corp.1 and University of Wisconsin School of medicine and Public Health2, Madison, WI
1. Abstract
4. Kinase Activity By Monitoring ADP Produced
(ADP-Glo™ Assay)
ADP conversion curves at different
ADP/ATP concentrations
Adenine nucleotides are major determinants of the energy status of
the cell and thus any modulation of their cellular concentration has
significant consequences to cellular metabolism, cellular growth and
cell death. ATP generating enzymes are usually involved in anabolic
processes while ATP consuming enzymes are involved in catabolite
processes. We have developed biochemical assays that monitor the
concentrations of ATP, ADP and AMP in a biochemical reaction. For
instance ATP depletion dependent assays and ADP generating assays
monitor the activity of kinases and ATPases. These universal assays
can measure the activity of various enzymes with no modification of
the native substrate and the ability to use diverse substrates such as
proteins, peptides, lipids, sugars, etc. The AMP generating reactions
such as ubiquitin ligases, aa aminoacyl t-RNA synthetases, DNA
ligases, cAMP-dependent phosphodiesterases, etc. can be also
monitored with high sensitivity and reproducibility. We will show data
using these various technologies in monitoring the various adenine
nucleotide concentrations in biochemical reactions.
10. Z’ Values, %CV, and S/B for the 16 Plates
(AMP-Glo™ Assay)
7. Quantification of AMP in E3 Ub. Ligase Reaction
05.17
E3 Titration with 0.04µg Ubiquitin-WT with 100µM
ATP Using AMP-Glo™
Ubiquitin Ligase (2µM, 1Hr Rxn) Study with 100µM ATP Using
AMP-Glo™
2.0E+06
R² = 0.9973
1.5E+06
RLU
DRLU
6.0E+04
4.0E+04
1.0E+06
2.0E+04
5.0E+05
0.0E+00
0.0E+00
0
100
200
300
400
1.5µM AMP
Rxn Buffer
Average
StDev
%CV
Average
StDev
%CV
S/B ratio
Z'
Pl.1
378,510
15,181
4.0
594
617
103.7
637
0.875
Pl.2
369,828
17,942
4.9
821
757
92.3
451
0.848
Pl.3
363,068
18,639
5.1
783
756
96.6
464
0.839
Pl.4
367,053
16,428
4.5
832
816
98.0
441
0.859
Pl.5
361,599
19,385
5.4
855
811
94.9
423
0.832
Pl.6
356,236
14,775
4.1
720
723
100.3
495
0.869
Pl.7
347,899
16,438
4.7
704
701
99.6
494
0.852
Pl.8
340,925
16,976
5.0
564
554
98.2
604
0.845
Pl.9
347,537
16,596
4.8
762
761
99.8
456
0.850
Pl.10
344,758
15,765
4.6
884
829
93.8
390
0.855
Pl.11
343,265
17,694
5.2
570
593
104.0
602
0.840
Pl.12
335,801
18,563
5.5
684
674
98.6
491
0.828
Pl.13
337,908
17,757
5.3
720
690
95.9
470
0.836
Pl.14
334,863
16,817
5.0
691
685
99.2
484
0.843
Pl.15
334,951
17,507
5.2
649
667
102.8
516
0.837
Pl.16
331,386
15,516
4.7
638
628
98.4
519
0.854
68,184
78
2,834
1576
704
99
496
0.848
500
E3 (nM)
ADP-Glo™ measures ADP produced in a kinase Reaction
Sum=
Average
2. Quantification of Cellular ATP
(Promega Kinase-Glo®)
"Negative" Ctrl.
AMP-Glo assay
8.0E+04
2.5E+06
"Positive" Controls
5. Quantification of AMP in Biochemical Reaction
(DNA Ligase – T4, ATP substrate)
349,724
8. Quantification of AMP in cAMP-PDE Reaction
(cAMP-Phosphodiesterase PDE4B2)
16,999
4.9
T4 DNA Ligase Titration with 0.5µg DNA
(pBR322)/Rxn with 100µM ATP Using AMP-Glo™
y = 5E+06x + 410472
R² = 0.9986
(in Buffer 1)
Using K-R Buffer
120
3.0E+05
2.0E+07
y = 3E+06x + 376284
R² = 0.9959
(in BUffer 2)
1.0E+07
100
2.5E+05
R² = 0.9984
Activity %
RLU
AMP-Glo™ vs. LOPAC
Using Buffers
3.0E+07
0.0E+00
2
4
6
8
2.0E+05
10
DRLU
0
ATP, µM
ATP Level in HEK293 & CHO Using Kinase-Glo®
7.00
10K
5K
1.5E+05
80
60
LOPAC compounds
+/- 3 St.Dev.
+/- 25%
2.5K
1.0E+05
40
6.00
5.00
mM
717
11. LOPAC Library Screen Results
(AMP-GloTM Assay)
ATP Standard Titration Using Kinase-Glo®
with Different Buffers
4.0E+07
Sum=
5.0E+04
20
4.00
0.0E+00
3.00
0
2
4
6
8
10
12
0
T4 Ligase, U/Rxn
2.00
0
200
400
600
800
1000
1200
Compound serial #
1.00
0.00
CHO
HEK293
3. Kinase Activity using ATP Depletion
(Promega Kinase-Glo®)
6. Quantification of AMP in Biochemical Reaction
(DNA Ligase – E. coli, NAD substrate)
9. Quantification of Cellular ADP & ATP
(CHO & HEK293)
ATP & ADP Level in HEK293 & CHO Using AMP-Glo™
E. coli DNA Ligase Titration with 10µM Oligos & 10µM
NAD Using AMP-Glo™
7.00
200,000
120,000
R² = 0.9919
NADP + Oligos + Ligase (20min Rxn)
RLU
140,000
80,000
60,000
NADPH + Oligos + Ligase (20min Rxn)
120,000
•Luminescent, Free of fluorescence interference
5.00
•HTS Formatted (96-, 384-, and 1536-well Plates)
4.00
R² = 0.998
NAD + Oligos + Ligase (20min Rxn)
100,000
80,000
3.00
60,000
40,000
•Universal: AMP and ADP Detection in the Presence or
Absence of ATP as Substrate
•Homogenous, Nonradioactive and Antibody free
ADP (mM)
NADH + Oligos + Ligase (20min Rxn)
160,000
100,000
ATP (mM)
6.00
NAD + Oligos + Ligase (20min Rxn)
180,000
mM
140,000
RLU
8.00
Nicodinamide specificity as Substrate in E. coli
DNA Ligase (10µM Oligos Using AMP-Glo™)
12. Features of Promega Luminescent
GloTM Technologies
•Robust Assays (Z’>0.8)
2.00
40,000
20,000
20,000
0
1.00
0
0
0.5
1
1.5
2
E. coli DNA Ligase, U/Rxn/5µl
2.5
3
0
2
4
6
8
10
12
•Ultrasensitive (pmoles of nucleotides)
Nicodinamides (µM)
0.00
HEK293 (10K cells/well)
HEK293 (25K cells/well)
CHO (10K cells/well)
CHO (25K cells/well)
•Stable Signal
Kinase-Glo® measures the remaining ATP after a kinase Reaction
www.promega.com
For technical information: Said.Goueli@Promega.com