Download HTRF phospho-IKKβ is more sensitive than Western-Blot

Do all cell-based kinase assays perform similarly?
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A side-by-side comparison of HTRF , Western Blot, ELISA and
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AlphaScreen SureFire
F. Charrier-Savournin, J. Vallaghé, N.Costy, S.Douzon, C.Chouvet, C.Boisseau, F. Degorce, E. Trinquet
Cisbio Bioassays, Codolet, France
Assays workflows
phosphorylated, a time-resolved FRET process will occur
between the 2 labeled antibodies (scheme 1: assay principle &
protocol). In this study, the performances of different HTRF
cellular kinase assays were benchmarked against different
technologies commonly used in the kinase research field, such
as Western Blot, Elisa and SureFire.
This poster provides comparative data on the following assays:
p-IKKb, p-Stat3, p-p38 MAPK, p-mTor and p-Creb, with a
particular emphasis on p-IKKb.
HTRF
Surefire
Day 1
Functional responses induced by pharmacological compounds
can be assessed through the quantification of specific biomarkers.
Among them, protein phosphorylation is a ubiquitous readout
reporting for various biological responses such as mitogenstimulated proliferative effect and inflammatory response upon
cytokine exposure, as well as metabolic response following
hormonal treatment. HTRF cellular kinase assays are intended
to measure the phosphorylated status of a protein in a cellular
context. The detection is achieved using a pair of antibodies
labeled with HTRF probes: a first antibody raised against the
phosphorylation residue of interest, and the second raised
against the total protein. If the protein of interest becomes
ELISA
Western blot
Sead cells (in micro plates or flask)
Lyse cells
Day 2
Introduction
transfer cell lysate to 384-sv plate
Add HTRF® conjugates
Incubate from 2 hours up to overnight
Read on compatible fluorescence reader
transfer cell lysate to 384-sv plate proxyplate transfer cell lysate to precoted 96-well plate
Add acceptor beads
Incubate for ON ay 4°C
Incubate for 2 hours
Add light sensitive donor beads
Incubate for 2 hours in the dark
Read on compatible fluorescence reader
Day 3
Assay principle and protocol
Wash 4 times
Add detection antibody
Incubate for 1 hour at 37°C
Wash 4 times
Add HRP substrate
Incubate for 10 min at 37°C
Add stop reaction
Lyse cells
Dilute cell lysates in laemly sample buffer
SDS page
Gel electrotransfert
Amido black coloration on the membrane
Read on spectrophotometer
Day 4
Saturate with BSA the membrane
Incubate for 1h at Rt
Add primary specific antibody
Incubate for 2h at Rt
Wash 3-times in TBS-T
Add HRP-conjugates secondary antibody
Incubate for 1h at Rt
Wash 3-times in TBS-T
Add chemiluminescent HRP-substrate
Transfer
16µL
Supernatant
Read on a CCD imaging system
50µL cells
Read on an HTRF
compatible reader
+50µL
compounds
Discard
supernatant
Lysis
HTRF and ELISA phospho-IKKb assays perform similarly
Detection of phospho-proteins
Method
5000
HTRF protocol: as described in Scheme 1, with 25µl of lysis buffer.
AlphaScreen® SureFire protocol: as per supplier’s instructions
(AlphaScreen® SureFire® IKKβ (p-Ser177/181) Assay Kits (TGRKBS500).
In this case, 25µl of aggressive lysis buffer were used and experiments
were performed under low-light conditions.
Ratio HTRF
(PheraFS lamp 2h)
Stimulation: IL1b for 15 min.
4000
S/B
4.2
1.4
4000
3000
1000
-13
-12
-11
-10
-9
log [IL1β ] M
P-IKKβ 87kDa
P-IKKα 85kDa
185000 92500 46200 2313
Ratio HTRF
(PheraFS lamp 2h)
Detection Protocol: 16µl of serial dilutions of cellular
lysates (as indicated in cells/well) on the figure were
either analyzed by HTRF or subjected to SDS-PAGE
and Western Blot procedure.
0.8
3.6
3000
0.6
2000
0.4
1000
0.2
0
-14
-13
-12
-11
-10
-9
-8
0.0
log [IL1β ] M
1156
580
289
145
72
Cellular kinase assay
Cell line model
Reference compound
Cellular kinase assay
HTRF
Western-blot
P-STAT3 (Y705)
Hela
IFNa
P-STAT3
750
750
P-p38 MAPK (T180-Y182)
Hela
anisomycin
P-p38 MAPK
1250
10000
P-IKKβ (Ser177/181)
Hela
IL1b
P-IKKβ
580
46500
P-mTOR (S2448)
HEK293
rapamycin
P-mTOR
1250
1250
P-CREB (S133)
HEK293
Forskolin
P-CREB
6250
6250
P-MEK1 (S217/S222)
Hela
EGF
P-MEK1
600
2400
0
Assay Window Comparison
5000
4000
3000
S/B 5.8
2000
1000
0
as-
HTRF vs WB:
Sensitivity comparison (cells / well)
Experimental conditions
Number of cells
Lysis : After cell culture medium removal, cells were
washed with PBS was performed using 3ml of HTRF
lysis buffer for 45 min. After 10min centrifuging at
2400rcf, cell lysate supernatant was collected.
3.5
Performance of HTRF cellular kinase
says refered to other technologies
-8
Method
Stimulation : 0.5nM of IL1b for 15 min.
ELISA protocol: as per supplier’s instructions (PathScan® PhosphoIKKb (Ser177/181) Sandwich ELISA Kit #7080).
S/B
2000
HTRF phospho-IKKb is more sensitive than
Western-Blot
Plating : 5 million Hela cells were seeded in as flask
(T175cm2) and incubated for 72h at 37°C under 5%
CO2.
4000
1.0
HTRF
ELISA
EC50 2.48 10 -11 3.58 10 -11
3000
2000
0
-14
Plating: Plating:100 000 Hela cells per well in a 96 well plate were
seeded, then incubated for 24h at 37°C under 5% CO2
HTRF protocol: as described in Scheme 1, with 50µl of lysis buffer.
Surefire signal
(Envision lamp 2h)
Plating: Plating:80 000 Hela cells per well in a 96 well plate were
seeded, then incubated for 24h at 37°C under 5% CO2.
5000
Stimulation: IL1b for 15 min.
5000
HTRF Surefire
EC50 1.06E-11 1.79E-11
Method
DO 450nm
HTRF phospho-IKKb shows superior
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performance than AlphaScreen SureFire
Ratio HTRF
(PheraFS lamp ON)
24h
Pharmacological parameter (EC50 in M)
Cellular kinase
HTRF
SureFire®
ELISA
Cellular kinase
HTRF
SureFire®
ELISA
P-STAT3
7.7
3
5.3
P-STAT3
10.7E-10
1.3E-9
5.9E-10
P-p38 MAPK
10.8
13.5
3.5
P-p38 MAPK
7.6E-8
6.1E-8
8.7E-8
P-IKKβ
3.5
1.4
3.6
P-IKKβ
2.48E-11
1.79E-11
3.58E-11
P-mTOR
2.4
1
2.2
P-mTOR
5.5E-6
-
4.7E-6
P-CREB
4.1
2.5
4.6
P-CREB
1.4E-6
3.6E-6
1.1E-6
P-MEK1
3.4
nd
2.8
P-MEK1
7E-10
nd
1E-9
Conclusion
The HTRF cellular kinase platform has been optimized for the investigation of site-specific phosphorylation of endogenous
proteins in whole cells. With 12 phosphospecific assays, it covers key targets implicated in diverse signaling pathways, and
enables an accurate and sensitive determination of phosphorylation events in whole cells using the same technology. In this
study, a panel of HTRF cellular kinase assays was compared to other widely used technologies. Experiments were performed
strictly according to the different suppliers’ instructions.
Overall, HTRF cellular kinase assays perform equally well or better than the alternative methods:
• Out of 6 assays tested, 5 showed a significantly better assay window when compared to AlphaScreen, in particular for
p-IKKB, p-STAT3 and p-mTOR.
• All assays performed similarly to or better than their equivalent ELISA.
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HTRF Symposium - 04/2013
Copyright © 2013 Cisbio Bioassays, France. HTRF®, TRACE®, and the HTRF™ logo are trademarks belonging to Cisbio Bioassays.
• 3 assays were clearly more sensitive than Western Blot, enabling a significant improvement in terms of cell consumption.
These HTRF cellular kinase assay kits have been optimized in order to enable a rapid, mix-and-read (no washing steps), nonradioactive, and miniaturizable assessment of phosphoproteins.
The study also shows that not only do these assays display superior output when compared to other well-established technologies,
but they also provide a streamlined approach, with a minimum of assay steps, and thereby constitute a very cost-effective
technological solution when undertaking scientific projects using a kinase readout, or when investigating these enzymes as
drug targets.
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