Procarta™ Transcription Factor Assay

Procarta™ Transcription Factor Assay:
High Throughput, Quantitative Platform for the
Measurement of Activated Transcription Factors
Takuro Yaoi, Xin Jiang, Sherry Huang, Aiguo Zhang, & Brendan Yee
Presented at the 4th Annual
Planet xMAP® USA Symposium
March 12-14, 2007
Dana Point, CA
Procarta™ Transcription Factor Assay
the Measurement of Activated Tran
Takuro Yaoi, Xin Jiang, Sherry Huang, Aiguo Zhang, & Brendan Yee
Abstract
Transcription factors (TFs) play a crucial role in the
regulation of gene expression in the human genome
and are highly regulated by a variety of mechanisms. A
single extracellular stimulus can trigger multiple signaling pathways, and these in turn can activate multiple
TFs to mediate the inducible expression of target genes.
Alterations in the activities of TFs are often associated
with human diseases, such as altered AP1, ER, and p53
function in cancer, NFκB in inflammatory diseases, and
PPARγ in obesity. A systematic assay for profiling the activation of TFs will aid in elucidating the mechanisms of
TF activation, reveal altered TFs associated with human
diseases, and aid in developing assays for drug discovery.
Here, we describe a commercially available 40-plex
Luminex® based assay for the measurement of activated
TFs. This assay is designed around the standard 96-well
plate format enabling high-throughput, multiplex
profiling of the DNA binding activity of TFs in multiple
samples with high-sensitivity.
Introduction
A single signal pathway can activate multiple
Transcription Factors, and a single TF can be activated
by multiple signal pathways. For example, TNFα can
activate NFκB and AP1 as well as other TFs such as E47.
NFκB can be activated by the cytokines TNFα, IL1, and
the phorbol ester PMA. However, accurate elucidation of
these complex, interconnected relationships between
signaling molecules requires a technique that can profile
the activities of multiple TFs simultaneously. In vitro
analysis of TF’s has been historically conducted using the
electrophretic mobility shift assay (EMSA) and for in vivo
analysis through the use of a luciferase reporter assay.
Both of these assays, however, are single plex assays,
which detect only one TF per reaction and, therefore, are
not suited for high throughput profiling of multiple TFs
with multiple samples. At Panomics, we have developed
a commerically available, multiplex assay that can analyze
up to 40 TFs from a nuclear extract or whole cell lysate
sample and up to 96 samples/controls in a micro­titerplate
based format utilizing the Luminex technology. This 40
plex TF assay is a sensitive and quantitative method for
profiling the activation of multiple TFs.
Methods
Format
Number of Target TF
per Reaction
Number of Samples
per Assay
EMSA
Gel Electrophoresis
1
1 per 3 Lane
ELISA
Antibody Detection
1
96 per Plate
Protein/DNA Array
DNA Based Membrane
50–345
1 per Membrane
Microsphere-based Assay
Multiplexed Microsphere
25–50
96 per Plate
Assay Overview
The Procarta Transcription Factor Assay is designed to
measure the activated transcription factors through the
use of double stranded cis-elements (the sense strand is
biotinylated). A mixture of up to 40 different cis elements
are incubated with individual samples prepared from
either whole cell or nuclear extracts. The samples are
then transferred to a separation plate and the excess cis
elements and unbound proteins are removed through
the porous bottom of the separation plate.
Step 1:
Incubation of the cis
element probes with
the nuclear extract
or whole cell lysate in
96 well plate.
Once washed, the activated TF’s bound to the cis
elements are denatured; the proteins remain bound
the porous membrane and the cis elements are eluted
into a PCR plate. The cis elements are denatured at 95°C,
chilled on ice, then bound to Luminex beads conjugated
to the anti-sense sequence of the TF of interest. The
beads are wahsed and incubated with streptavidin-PE.
After incubation, the beads are then washed and read
on the Luminex platform.
Cis1
Incubation
Cisn
Cis2
Cisn
Step 2:
Transfer probe TF mixture to separation
plate and wash TF bound probe mix.
Denature cis elements away from TFs.
TFn
TF2
TF1
Cis2
Cis1
Separation
Step 3:
Denature cis elements by heat
and aneal to Luminex beads with
anti-sense sequence.
Cis1
Cisn
Cis2
Cis1
Luminex Assay
bead
1
Cis2
bead
2
Cisn
Step 4:
Add streptavidin PE to sample and
read on Luminex machine
bead
3
y: High Throughput, Quantitative Platform for
nscription Factors
Material and Methods
TF Plex 40-plex assay: Nuclear Extract from HeLa +/- PMA
RUNX/AML
CREB
GR/PR
NFAT
PAX3
AP1
E2F1
HIF-1
NF-E1(YY1)
PAX5
AP2
ELK1
HNF1
NF-E2
PPAR
AR
ER
IRF1
NFκB1
SMAD
ATF2
ETS/PEA
ISRE
NKX-2.5
STAT1
BRN3
FAST1
MEF-2
NF-Y
STAT3
CEBP
FKHR-1
MYOD
OCT
STAT4
C-MYB
GATA-1
NF-1
P53
STAT5
Probe only
Nuclear Extract –PMA
Nuclear Extract +PMA
12000
10000
MFI
8000
6000
4000
2000
STAT4
STAT5
STAT1
STAT3
PPAR
SMAD
PAX5
P53
PAX3
NKX-2.5
OCT
NF-Y
NF-E2
NFKB1
NF-E1 (YY1)
NF-1
NFAT
MYOD
MEF-2
IRF1
ISRE
HIF-1
HNF1
GR/PR
GATA-1
FAST1
FKHR-1
ER
ETS/PEA
ELK1
CREB
E2F1-1
CEBP
C-MYB
BRN3
AR
ATF2
AP2
AP1
0
RUNX/AML
Preparation of nuclear extracts and gel shift assays
(EMSA) were performed using kits (Panomics) according
to the manufacture’s instructions. For EMSA assays, 5 µg
of nuclear extract was used per reaction. The Procarta
Transcription Factor assay performed in this study was
carried out following the procedure in the Procarta TF
Kit User manual (Panomics). For Procarta assays, 2 µg of
nuclear extract was used per multiplex reaction. The
probes for the EMSA assay were 5’-biotin-labeled and
identical in sequences to the probes used in the Procarta
TF assay. A table and chart is illustrated below of the possible TF’s avaiable for profiling with the Procarta TF assay
as well as the specificity of the probes used in this assay.
14000
EMSA Confirmation
PMA Induction
The Procarta TF assay was confirmed using the electrophoretic mobility shift assay of selected transcription
factors. Results from EMSA gave similar fold indunction
compared to Procarta TF assay. Lane 1 is probe only,
Lane 2 is untreated, Lane 3 is treated at 30 min and Lane
4 is cold and labeled probes
Nuclear extracts from HeLa cells were prepared as
previously described but induced with PMA at 10 ng/ml.
The median fluorescent intensity was measured of
treated and untreated samples and displayed in the
graph above.
PPAR
AP2
1 2 3 4 1 2 3 4
CREB
NFkB
AP1
ER
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
EMSA Confirmation
Confirmation of the the PMA treated HeLa cells measured
by the Procarta TF assay was confirmed using the electrophoretic mobility shift assay of selected transcription
factors. Results from EMSA gave similar fold indunction
compared to Procarta TF assay. Lane 1 is probe only, Lane
2 is untreated and Lane 3 is PMA treated.
30000
25000
Nkx-2.5
STAT4
Stat1
Pax3
p53
NFkB -1
NF-E1(YY1)
NF-1
MEF-2
IRF1
HIF-1
GATA
ETS/PEA
Elk1
E2F1
c-myb
CEBP 10 nM
ATF2
AP2
RUNX/AML
TNFα Induction
15 min
/PR
ME
F2
NF
- E2
p5
3
5 min
30 min
GR
10 min
Et s
2.5 min
AP
2
CR
EB
NF
-E1
(Y Y
1)
NF
AT
PP
AR
Sm
ad
Sta
t3
Sta
t4
Sta
t5
AP
1
E2
F-1
ER
9
8
7
6
5
4
3
2
1
0
NF
kB
Fold Induction
7.00
6.00
5.00
4.00
3.00
2.00
1.00
NF
-E
2
p5
3
St
at
1
0.00
2
Fold Induction
Human HeLa cells were maintained in DMEM containing
10% fetal bovine serum, 100 units/ml penicillin and 0.1
mg/ml streptomycin at 37°C and 5% CO2. For nuclear
extraction of TNFα-treated HeLa cells, the cells were
starved for 16 h with DMEM containing 0.2 % FBS, 100
units/ml penicillin and 0.1 mg/ml streptomycin at 37°C
and 5% CO2, and treated with 10 ng/ml TNFα. Nuclear
extractions were prepared at time points of 2.5, 5, 10, 15
and 30 minutes induction. A 19 plex assay was performed
on these samples and fold induction was measured
by normalizing the treated samples agasint the
untreated sample.
EF
0
RUNX/AML (44)
AP1-1 (35)
AP2-1 (21)
AR (33)
ATF2 (26)
NF-Y (47)
CEBP (46)
FAST1 (12)
C-MYB (51)
CREB (52)
E2F1-1 (36)
EGR (24)
ELK1 (7)
ER (53)
ETS/PEA (34)
FKHR-1 (56)
GATA-1 (61)
GR/PR (54)
HIF-1(2) (55)
HNF1 (64)
IRF1 (62)
ISRE (28)
MEF-2 (73)
MYOD (37)
NF-1 (27)
NFATC (76)
NF-E1(YY1) (25)
NF-E2 (20)
NFKB1 (11)
OCT (17)
P53 (18)
PPAR (42)
PAX3 (19)
SMAD (43)
STAT1 (63)
STAT3 (41)
STAT4 (65)
STAT5 (66)
NKX-2.5 (32)
TFIID (45)
5000
GR
10000
Cos-1 cells were maintained in DMEM containing 10%
fetal bovine serum, 100 units/ml penicillin and 0.1 mg/ml
streptomycin at 37°C and 5% CO2. Cos-1 cells were transfected with pCMV-GRα expression vector (Panomics) and
the cells were seeded without antibiotics sixteen hours
prior to transfection. Transfection was performed using
LipofectAMINE 2000 reagent. Cos-1 cells were plated in
10 cm culture dishes and transfected. After 16 h, the cells
were treated with or without dexamethasone for one
hour and then the nuclear extracts were prepared. A 19
plex assay was performed on the treated and untreated
samples. Fold induction was measured by normalizing
the treated samples agasint the untreated sample.
M
15000
kB
Et
s
AP
2
CR
NF
E
-E B
1(
YY
1
NF )
AT
c
PP
AR
Sm
ad
St
at
3
St
at
4
St
at
5
AP
1
E2
F1
ER
20000
1 2 3
1 2 3
1 2 3
1 2 3
AP1
NF-1
NF-E2
STAT4
GR Detection
IRF1
ISRE
MEF-2
MyoD
NF-1
NFATc
NF-E1(YY1)
NF-E2
NFkB -1
Oct
p53
PPAR
Pax3
Smad
Stat1
STAT3
STAT4
Stat 5
Nkx-2.5
TFIID
NF
RUNX/AML
AP1
AP2
AR
ATF2
NF-Y
CEBP 10 nM
FAST1
c-myb
CREB
E2F1
EGR
Elk1
ER
ETS/PEA
FKHR
GATA
GR/PR
HIF-1
HNF1
The Procarta TF assay is a high throughput assay
platform for the quantitative measurement of up to
40 different activated TF’s from a single sample. The
throughput and ease of use of this assay lends itself
to being an exceptional profiling tool for biomarker
discovery and validation.
• Insight: More information to better understand
signaling pathways
GR EMSA Confirmation
Nuclear extracts of transfected and nontransfected Cos-1 cells were prepared
as previously described and measured
for the GR transcription factor. Lane 1
is probe only, Lane 2 is untransfected,
and Lane 3 is transfected. Results match
change of fold induction to that of
Procarta TF Plex assay.
Summary
• High throughput: 96 samples with 40 TF’s per sample
• Accurate: Quantitation utilizing Luminex bead
technology
• Sensitive: Assay uses 2 µg of nuclear extract
• Easy to Use: ELISA like workflow and data under
4 hours
1 2 3
© 2007 Panomics, Inc. All rights reserved.
Abstract
Transcription factors (TFs) play a crucial role in the regulation of gene expression in the human
genome and are highly regulated by a variety of mechanisms. A single extracellular stimulus
can trigger multiple signaling pathways, and these in turn can activate multiple TFs to mediate
the inducible expression of target genes. Alterations in the activities of TFs are often associated
with human diseases, such as altered AP1, ER, and p53 function in cancer, NFκB in inflammatory
diseases, and PPARγ in obesity. A systematic assay for profiling the activation of TFs will aid in
elucidating the mechanisms of TF activation, reveal altered TFs associated with human diseases,
and aid in developing assays for drug discovery. Here, we describe a commercially available
40-plex Luminex® based assay for the measurement of activated TFs. This assay is designed
around the standard 96-well plate format enabling high-throughput, multiplex profiling of the
DNA binding activity of TFs in multiple samples with high-sensitivity.
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© 2007 Panomics, Inc. All rights reserved. Procarta is a trademark of Panomics, Inc. Luminex and xMAP are registered trademarks of Luminex Corp. All other trademarks are the property of their respective owners.