One-Step TF Activation Reporter Array I Signosis

Signosis
Innovative Plate Assay Solutions
One-Step TF Activation Reporter Array I
(For Research Use Only)
Catalog #: BA-0001 and BA-0002
Introduction
Transcription factors (TFs) are the downstream
effectors of intracellular signaling pathways. The cellbased reporter assay is often used to monitor changes
in TF activation. Measuring the activation of multiple
TFs simultaneously can decipher a clear picture of the
biological status of signaling pathways in cells.
Signosis has developed One-Step TF Activation
Reporter Array I, which can analyze 22 most popular
TFs in living cells in response to different stimuli in a
high throughput workflow. The individual TF
reagents are arranged in 3X8 format in a pre-coated
sterile 96-well white plate. You just need to add your
cells and treat with the stimuli of your interest without
any usage of transfection reagents. The white plate is
ready for luciferase reading in a luminometer.
Handling upon arrival
It is strongly recommended that you store TF
Activation Reporter Array I at 4°C in the dark as soon
as possible upon arrival. Do not store in -20°C.
IMPORTANT: Baculovirus are light-sensitive and
require gentle handling. Avoid vortexing and frequent
pipetting as mechanical forces can damage the virus
and decrease the viral titer.
NOTE: Shelf life is 3 months in the dark at 4ºC.
Use all wells at the same time and do not save some
for a later time.
Materials provided
Principle
This array utilizes 24 recombinant viruses that are
generated from insect cells with a modified
baculovirus system that contains mammalian delivery
element, TF consensus sequence and luciferase
reporter gene. The recombinant viruses are pre-coated
in 96-well sterile white plate to simplify the
assay. Experimentally, seed the cells overnight in the
96-well plate at 50%-70% confluency. Then treat
with your stimuli, followed by luciferase assay. The
induction of luciferase activities has been
demonstrated in a wide range of cell lines, including,
HEK293, HeLa, HepG2, MCF7 and primary fibroblast
cells with matching TF activators. Most importantly,
this array can be used in routine lab settings without
additional or special safety protocols as these
recombinant viruses do not infect or replicate and are
easily inactivated with 70% ethanol in case of a spill.
BA-0001 - One pre-coated sterile 96-well white plate
BA-0002 - Five pre-coated sterile 96-well white plates
Materials required but not provided
(May be substituted with a comparable third-party
product)


Luciferase assay -- Promega P/N E-1500
Passive lysis buffer -- Promega P/N E-1941
Signosis, Inc. • 1700 Wyatt Drive Suite 10-12 • Santa Clara, CA 95054 • Tel 408 747 0771 • Fax 408 864 2182 •
[email protected]
Assay procedure for adherent cells
List of TF Reporters:
NOTE: Before removing the foil in the tissue culture
hood, centrifuge the 96-well plate at 1,000 RPM for
30 seconds in a swinging-bucket rotor. Make sure to
balance the plate with a similar counterweight or
the plate and centrifuge may sustain damage.
1.
NFkB : NFκB pathway.
NFAT : Calcium signaling.
TCF/LEF : Wnt/β-catenin pathway.
ATF6 : Unfolded protein response/ ER stress.
GAS/ISRE : JAK/STAT pathway.
ER : Estrogen receptor pathway.
AP1 : JNK/MAPK pathway.
SMAD : TGFβ-SMAD pathway.
STAT1 : JAK/STAT pathway.
YY1 : Yin Yang 1 transcription factor.
HSF : Heat shock response.
NRF2 : Antioxidant response pathway.
XBP1 : Unfolded protein response/ ER stress.
PPARg: PPARγ, fatty acid catabolism.
HIF1 : Hypoxia response pathway.
AR : Androgen receptor pathway.
IRF : Interferon regulatory factor.
SP1 : Specificity protein 1 transcription factor.
PR : Progesterone receptor pathway.
CREB : PKA-cAMP pathway.
C/EBP : CCAAT-enhancer-binding proteins.
SRF : Serum response pathway.
Gli : Gli-hedgehog pathway.
Positive Control : CMV Promoter-luciferase.
The day before performing the assay, trypsinize
the cells and seed 100uL per well of the precoated 96-well sterile white-wall plate at 50-70%
confluency.
Note: 5,000 to 10,000 cells per 100uL yields
approximately 50-70% confluency for most
cultured cell lines.
Please empirically
determine the cell density for your cells of
interest before each experiment.
2.
Incubate the plate in a humidified incubator at
37oC with 5% CO2 overnight to 24 hours. No
media change necessary.
Add appropriate treatment to cells to induce
luciferase activity.
Remove the media by aspiration and add 100l
of PBS to each well.
Remove PBS by aspiration and add 20l of
passive lysis buffer to each well.
Incubate cells in lysis buffer for 15 minutes at
room temperature.
Add 100l of luciferase substrate to each well
and gently pipette up and down.
Immediately read the plate in a luminometer.
3.
4.
5.
6.
7.
8.
Diagram of One-Step TF Activation Reporter
Array I
A
B
C
D
E
F
G
H
1
NFkB
NFAT
TCF/LEF
ATF6
GAS/ISRE
ER
AP1
SMAD
2
STAT1
YY1
HSF
NRF2
XBP1
PPARg
HIF1
AR
3
IRF
SP1
PR
CREB
C/EBP
SRF
Gli
CMV
4
NFkB
NFAT
TCF/LEF
ATF6
GAS/ISRE
ER
AP1
SMAD
5
STAT1
YY1
HSF
NRF2
XBP1
PPARg
HIF1
AR
6
IRF
SP1
PR
CREB
C/EBP
SRF
Gli
CMV
7
NFkB
NFAT
TCF/LEF
ATF6
GAS/ISRE
ER
AP1
SMAD
8
STAT1
YY1
HSF
NRF2
XBP1
PPARg
HIF1
AR
9
IRF
SP1
PR
CREB
C/EBP
SRF
Gli
CMV
10
NFkB
NFAT
TCF/LEF
ATF6
GAS/ISRE
ER
AP1
SMAD
11
STAT1
YY1
HSF
NRF2
XBP1
PPARg
HIF1
AR
Signosis, Inc. • 1700 Wyatt Drive Suite 10-12 • Santa Clara, CA 95054 • Tel 408 747 0771 • Fax 408 864 2182 •
[email protected]
12
IRF
SP1
PR
CREB
C/EBP
SRF
Gli
CMV

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