TransAM™
ATF-2
Transcription Factor Assay Kits

(version B)

Catalog Nos. 42396 & 42896)

Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, California 92008, USA
Toll free:877 222 9543
Telephone:
760 431 1263
Fax:
760 431 1351
Active Motif Europe
104 Avenue Franklin Roosevelt
B-1330 Rixensart, Belgium
UK Free Phone: 0800 169 31 47
France Free Phone:
0800 90 99 79
Germany Free Phone: 0800 181 99 10
Telephone:		+32 (0)2 653 0001
Fax:		
+32 (0)2 653 0050
Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Telephone:+81 3 5225 3638
Fax:
+81 3 5261 8733
Copyright 2009 Active Motif, Inc.

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Information in this manual is subject to change without notice and does not constitute a commitment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any warranty of any
kind, either explicit or implied. Information may be changed or updated in this manual at any time.
This documentation may not be copied, transferred, reproduced, disclosed, or duplicated, inwhole or in part, without the prior written consent of Active Motif, Inc. This documentation is
proprietary information and protected by the copyright laws of the United States and international treaties.
The manufacturer of this documentation is Active Motif, Inc.
© 2009 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA 92008. All rights reserved.
All trademarks, trade names, service marks or logos referenced herein belong to their respective
companies.
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TABLE OF CONTENTS

Page

Overview  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Flow Chart of Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Introduction
MAPK Regulated Transcription Factors  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Transcription Factor Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
TransAM ATF-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Kit Performance and Benefits  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 6Kit Components and Storage
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Protocols
Buffer Preparation and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Quick Chart for Preparing Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  10
ATF-2 Transcription Factor Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  10
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Appendix
Section A. Preparation of Nuclear Extract  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section B. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Section C. Related Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  16
Technical Services  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

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Overview
The activation of mitogen-activated protein kinase (MAPK) signal transduction pathways is
responsible for the phosphorylation of transcription factors by the terminal kinase in these cascades. MAPK pathways play major roles in converting mitogenic and stress stimuli into nuclear
responses,1 and therefore, accurate monitoring of MAPK substrates in cells, tissues and animals is
crucial for many biomedical research and drug development projects. To date, such research projects are tedious, time consuming and lack high-throughput screening methods.
With its patented TransAM™ method*, Active Motif introduced the first ELISA-based kits to detectand quantify transcription factor activation. TransAM Kits combine a fast, user-friendly format
with a sensitive, specific assay. TransAM ATF-2 Kits are designed specifically for the study of the
MAPK regulated transcription factor ATF-2. The kits contain a 96-well plate to which oligonucleotide containing an ATF-2 consensus binding site has been immobilized. The activated ATF-2
contained in nuclear extracts specifically binds to this oligonucleotide. By using an antibody thatis directed against phosphorylated ATF-2, the activated complex bound to the oligonucleotide is
detected. Addition of a secondary antibody conjugated to horseradish peroxidase (HRP) provides
sensitive colorimetric readout that is easily quantified by spectrophotometry. The 96-well platewith detachable wells is suitable for manual use or high-throughput screening applications.
TransAM ATF-2 Kits are available in two sizes:

product

formatcatalog no.

TransAM™ ATF-2

1 x 96 rxns
5 x 96 rxns

42396
42896

Active Motif also offers TransAM MAPK Family Kit for the study of ATF-2, c-Jun, c-Myc, MEF2 and
STAT1, all of which are regulated by MAP kinase cascades. See these and other Active Motif products related to ATF-2 in Appendix, Section C.

* Technology covered by AAT-filed patents and licensed to Active Motif.

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1Flow Chart of Process

2

1

5

4

3

7

6

8

9

10

11

12

A
B
C
D
E
F

Cell extractcontaining activatedtranscription factor

G
H

Oligonucleotide
coated plate

Add cell
extract
21

11

01

9

8

7

6

5

4

3

2

1
A
B

C
D
E
F
G
H

Add primary
antibody

1 hr.
2

1

9

8

7

6

5

4

3

11

10

12

A
B
C
D
E
F
G
H

Add anti-IgG
HRP conjugate
21

11

01

9

8

7

6

5

1 hr.

4

3

2

1
A
B

C
D
E
F
G

Add developing
and stop solution

H

1 hr.
3

2

1

5

4

7

6

A
B
C
D
E
F
G
H

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8

9

10

11

12

Introduction
MAPK Regulated Transcription Factor
The transmission of extracellular signals into intracellular responses is a complex process that
often involves the activity of mitogen-activated protein kinases (MAPKs) (for review, see 2). The
MAPK pathway is a three kinase cascade consisting of a MAPK kinase (MAPKKK or MEKK) that
activates a MAP/ERK kinase (MEK or MAPKK). This stimulates a phosphorylation-dependent
increase in the activity of the MAP kinase. Upon activation, MAPKs phosphorylate a variety of
intracellular targets including transcription factors, transcription adaptor proteins, membrane andcytoplasmic substrates as well as other protein kinases, as illustrated below.2
MAP kinase

Activator

Nuclear Substrate

ERK1/2Growth factors
Serum
Hormones
Cytokines
Small molecules

ATF-2, Elk-1, c-Fos, c-Myc, SAPs,
c-Jun, NeuroD1, PDX-1, STAT3,
RSKs, Mnks, MSK

ERK5

Growth factors
Serum
Hormones
Osmotic stress

MEF2, RSKs

p38

Hormones
Cytokines
Osmotic stress
Heat shockATF-2, Elk-1, MEF2, SAPs, STAT1,
STAT3, MAPKAPs, Mnks, MSK

JNK

Hormones
Cytokines
DNA and protein synthesis inhibitors
Osmotic stress

ATF-2, c-Jun, Elk-1, STAT1, STAT3

At least three parallel MAPK pathways exist in humans. The extracellular signal-regulated protein
kinase (ERK) pathway primarily transmits mitogenic and differentiation stimuli, while the c-Jun
N-terminal kinase (JNK) and p38 pathways predominantly transmit stress and cytokine stimuli.1
c-Myc, an ERK substrate, is a transcription factor that regulates cell growth, cell differentiation,
glycolysis and apoptosis. Deregulation of c-Myc has been implicated in the origin of diverse humancancers. Elk-1 is a member of the ternary complex factor (TCF) sub-family of the ETS domain family.
Elk-1 can be stimulated by all three MAPK pathways, and its main function is the regulation of theactivity of the c-Fos promoter in response to extracellular stimuli.2 MEF2, a member of the MADSbox family, is mainly involved in muscle differentiation, but also plays roles in muscle hypertrophy,
neuronal survival and T-cell apoptosis.3 MEF2 is activated by both the p38 and ERK5 pathways.1
STAT1 is involved in activation of IFNα and γ genes, and is activated by p38 and JNK pathways.4
c-Jun is a member of the activator protein-1 (AP-1) family and is activated by both ERK1/2 and JNK
pathways.2 AP-1 members play roles in the expression of genes involved in proliferation and cell
cycle progression. ATF-2 is a member of the ATF/CREB family that binds to the cAMP response
element (CRE). ATF-2 is activated by ERK1/2, JNK and p38.2, 5
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3

Transcription Factor Assays
To date, three methods are widely used to measure MAPK regulated transcription factors such as
ATF-2, either directly or indirectly:1.

ATF-2 expression can be measured by Western blot, using antibodies raised against ATF-2.
This method is time consuming (up to 2 days once the cell extracts are prepared), and is notsuitable for processing large numbers of samples.

2.

The DNA-binding capacity of ATF-2 can be assayed by gel retardation, also called electrophoretic mobility shift assay (EMSA). In this method, cell extracts are incubated with aradioactive double-stranded oligonucleotide probe containing the consensus sequence for
ATF-2 binding. If ATF-2 is active in the cell extract, it will bind to the probe. Samples are thenresolved by electrophoresis on a native polyacrylamide gel, followed by autoradiography.
This method is sensitive, but like the previous procedure, it is time consuming (multiple daysof gel exposure may be required to achieve sufficient sensitivity) and it cannot be applied tohigh-throughput screening. Gelshift assays also require special precautions and equipment
for handling radioactivity.

3.

Another method used to assay ATF-2 activation is based on reporter genes, typicallyluciferase or β-galactosidase, placed under the control of a promoter containing the ATF-2
consensus sequence. This promoter can be artificial, made of several ATF-2 cis-elements anda TATA box, or natural. Limitations of this procedure are: (i) reporter gene assays have tobe repeated several times to obtain statistically reliable data; and (ii) reporter gene assays
are sensitive to confounding factors that may influence the expression level of the reporter
gene. Therefore, assays have to be carefully standardized. This method is sensitive and easy
to perform with a large number of samples but requires efficient cell transfection with thereporter plasmid.

TransAM ATF-2
Transcription factors that are activated by MAP kinases convert mitogenic and stress stimuli into
nuclear responses, and these diverse transcription factors play important roles in many areas ofcell proliferation and survival. However, this field has been hampered by the lack of convenient,high-throughput assays suitable for detection of transcription factors activated by MAPK.
To overcome this, Active Motif is introducing a high-throughput assay to quantify the MAPK regulated transcription factor ATF-2. The TransAM Kit combines a fast and user-friendly ELISA format
with a sensitive and specific assay for transcription factors. TransAM ATF-2 Kits contain a 96-wellplate to which an oligonucleotide containing the ATF-2 consensus site has been immobilized. The
active form of ATF-2 contained in the nuclear extract specifically binds to this oligonucleotide.
The primary antibodies used to detect ATF-2 will recognize an epitope on phosphorylated ATF-2
that is accessible only when ATF-2 is activated and bound to the target DNA. Utilization of asecondary HRP-conjugated antibody provides a sensitive colorimetric readout easily quantified byspectrophotometry. Once the nuclear extracts are prepared, this assay is completed in less than
3.5 hours. As this assay is performed in 96-well plates, a large number of samples can be handled
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4

simultaneously, enabling high-throughput automation. This assay is specific for ATF-2 activationand has been shown to be 10-fold more sensitive and 20-fold faster than the gel-retardation
technique. With the 3.5-hour TransAM procedure, we could detect activation using as little as 1 µgof nuclear extract. A comparable assay using EMSA required 10 µg of nuclear extract and a 3-day
autoradiography.
TransAM has many applications including the study of drug potency, inhibitor or activator proteins, and protein structure/function studies in the MAPK signaling pathway.

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5Kit Performance and Benefits
Detection limit: < 1 µg nuclear extract/well. TransAM ATF-2 is 10-fold more sensitive than EMSA.
Range of detection: TransAM provides quantitative results from 1 to 10 µg of nuclear extract/well.
Cross-reactivity: TransAM ATF-2 detects phosphorylated ATF-2 (Thr 71) from human origin. Reactivity with other species has not been determined.
Assay time: 3.5 hours.

1.2

1

ATF-2 activation (OD 450nm)

HeLa
HeLa + Anisomycin
0.8

0.6

0.4

0.2

0

0.625

1.25

2.5

5

10

Extract (µg/well)

Monitoring ATF-2 activation with the TransAM ATF-2 Kit:
Nuclear extracts from HeLa cells (Catalog No. 36010) and HeLa cells stimulated with anisomycin (Catalog No. 36111) wereassayed from 0.625-10 µg/well for ATF-2 activation using the TransAM ATF-2 Kit. Data shown are the results from wells
assayed in duplicate. These results are provided for demonstration only.

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6

Kit Components and Storage
TransAM ATF-2 Kits are for research use only. Not for use in diagnostic procedures. Except for the
nuclear extract that must be kept at -80ºC, kit components can be stored at -20ºC prior to firstuse. Then, we recommend storing each component at the temperature indicated in the tablebelow. All components are guaranteed stable for 6 months from date of receipt when stored
properly.
Quantity
1 plate / 5 plates

Storage

Phosphorylated ATF-2 antibody

11 µl / 55 µl

4°C for 6 months

Anti-mouse HRP-conjugated antibody

11 µl / 55 µl

4°C for 6 months

Wild-type oligonucleotide AM2

100 µl / 500 µl(10 pmol/µl)-20°C for 6 months

Mutated oligonucleotide AM2

100 µl / 500 µl(10 pmol/µl)-20°C for 6 months

HeLa (Anisomycin) nuclear extract

40 µl / 200 µl(2.5 µg/µl)-80°C for 6 months

Dithiothreitol (DTT) (1 M)

100 µl / 500 µl-20°C for 6 months

Protease Inhibitor Cocktail

100 µl / 500 µl-20°C for 6 months

Lysis Buffer AM1

10 ml / 50 ml

4°C for 6 months

Binding Buffer AM6

10 ml / 50 ml

4°C for 6 months

10X Wash Buffer AM2

22 ml / 110 ml

4°C for 6 months

10X Antibody Binding Buffer AM3

2.2 ml / 11 ml

4°C for 6 months

Developing Solution

11 ml / 55 ml

4°C for 6 months

Stop Solution

11 ml / 55 ml

4°C for 6 months

96-well ATF-2 assay plate

1/5

4°C for 6 months

Plate sealer

1/5

Room temperature

Reagents

Additional materials required• Multi-channel pipettor• Multi-channel pipettor reservoirs• Rocking platform• Nuclear extracts• Microplate spectrophotometer capable of reading at 450 nm (655 nm as an optionalreference wavelength)www.activemotif.com

7

Protocols
Buffer Preparation and Recommendations
Preparation of Complete Lysis Buffer
We provide an excess of Lysis Buffer AM1 in order to perform the assay AND to prepare customized nuclear extracts. Please refer to the Appendix Section A for a protocol to prepare a nuclear
extract. Our Nuclear Extract Kit can also be purchased separately (Cat. Nos. 40010 & 40410). Prepare the amount of Complete Lysis Buffer required for the assay by adding 1 µl of 1 M DTT and 10µl Protease Inhibitor Cocktail per ml of Lysis Buffer AM1 (see the Quick Chart for Preparing Buffers
in this section). Some of the protease inhibitors lose their activity after a few hours once diluted.
Therefore, we recommend adding protease inhibitors immediately prior to use. Any remaining
Complete Lysis Buffer should be discarded if not used in the same day.
Preparation of Complete Binding Buffer
Prepare the amount of Complete Binding Buffer AM6 required for the assay by adding 2 μl of DTT
per ml of Binding Buffer AM6 (see the Quick Chart for Preparing Buffers in this section). After use,
discard remaining Complete Binding Buffer.
Preparation of 1X Wash Buffer
Prepare the amount of 1X Wash Buffer required for the assay as follows: For every 100 ml of 1X
Wash Buffer required, dilute 10 ml 10X Wash Buffer AM2 with 90 ml distilled water (see the Quick
Chart for Preparing Buffers in this section). Mix gently to avoid foaming. The 1X Wash Buffer may
be stored at 4°C for one week. The Tween 20 contained in the 10X Wash Buffer AM2 may formclumps, therefore it is necessary to completely resuspend any precipitates by incubating at 50ºC
for 2 minutes and mixing prior to use.
Preparation of 1X Antibody Binding Buffer
Prepare the amount of 1X Antibody Binding Buffer required for the assay as follows: For every
10 ml of 1X Antibody Binding Buffer required, dilute 1 ml 10X Antibody Binding Buffer AM3 with 9
ml distilled water (see the Quick Chart for Preparing Buffers in this section)*. Mix gently to avoid
foaming. Discard remaining 1X Antibody Binding Buffer after use. The BSA contained in the 10X
Antibody Binding Buffer AM3 may form clumps, therefore it is necessary to completely resuspend
the precipitates by warming to room temperature and vortexing for 1 minute prior to use. Diluteboth primary and secondary antibodies to 1:1000 with the 1X Antibody Binding Buffer. Dependingon the particular assay, the signal:noise ratio may be optimized by using higher dilutions of both
antibodies. This may decrease the sensitivity of the assay.
* Volumes listed refer to the preparation of buffer for diluting both the primary & secondary
antibodies.

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8Developing Solution
The Developing Solution should be warmed to room temperature before use. The Developing
Solution is light sensitive, therefore, we recommend avoiding direct exposure to intense lightduring storage. The Developing Solution may develop a yellow hue over time. This does not affectproduct performance. A blue color present in the Developing Solution indicates that it has been
contaminated and must be discarded. Prior to use, place the Developing Solution at room temperature for at least 1 hour. Transfer the amount of Developing Solution required for the assay into
a secondary container before aliquoting into the wells (see the Quick Chart for Preparing Buffers
in this section). After use, discard remaining Developing Solution.
Stop Solution
Prior to use, transfer the amount of Stop Solution required for the assay into a secondary container (see the Quick Chart for Preparing Buffers in this section). After use, discard remaining Stop
Solution.
WARNING: The Stop Solution is corrosive. Wear personal protective equipment whenhandling, i.e. safety glasses, gloves and labcoat.
Nuclear extract
The HeLa (Anisomycin) nuclear extract is provided as a positive control for ATF-2 activation. This extract
is optimized to give a strong signal when used at 5 µg/well. We recommend aliquoting the extract in 5 µl
fractions and storing at -80ºC. Avoid multiple freeze/thaw cycles of the extract. Various cell extracts areavailable from Active Motif (see Appendix, Section C. Related Products).
Wild-type and mutated consensus oligonucleotides
The wild-type consensus oligonucleotide is provided as a competitor for ATF-2 binding in order tomonitor the specificity of the assay. This competition assay will confirm that the protein subunits
binding to the plate are specific for the ATF-2 consensus binding sequence. Used at 20 pmol/
well, the wild-type oligonucleotide will prevent ATF-2 binding to the probe immobilized on theplate. Conversely, the mutated consensus oligonucleotide should have no effect on ATF-2 binding.
Prepare the required amount of wild-type and/or mutated consensus oligonucleotide by adding
2 µl of appropriate oligonucleotide to 43 µl of Complete Binding Buffer per well being used (seethe Quick Chart for Preparing Buffers in this section). To allow for optimum competition, add
the oligonucleotide to the well first, then add the cell extract. It is not necessary to perform
an incubation step of the oligonucleotide in the well prior to addition of the cell extract. The
oligonucleotide competition only needs to be performed as a control. It is suggested to test theoligonucleotide competition each new cell type used.

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9Quick Chart for Preparing Buffers
Reagents to prepare

Components

For 1
well

For 1 strip(8 wells)

For 6 strips(48 wells)

For 12 strips(96 wells)

Complete Lysis Buffer

DTT
Protease Inhibitor Cocktail
Lysis Buffer AM1
TOTAL REQUIRED

0.01 µl0.12 µl11.12 µl11.25 µl

0.1 µl0.9 µl89.0 µl90.0 µl

0.6 µl5.4 µl534.0 µl
540.0 µl

1.2 µl10.8 µl
1.068 ml
1.08 ml

Complete Binding Buffer

DTT
Binding Buffer AM6
TOTAL REQUIRED

0.09 µl44.9 µl45 µl0.72 µl359.3 µl
360 µl4.32 µl2.15 ml
2.16 ml

8.64 µl
4.31 ml
4.32 ml

Complete Binding Buffer
with wild-type ormutated oligonucleotide

Wild-type or mutated oligo
Complete Binding Buffer
TOTAL REQUIRED

2 µl
43 µl
45 µl

16 µl
344 µl360 µl96 µl2.158 ml
2.16 ml

N/A
N/A
N/A

1X Wash Buffer

Distilled water
10X Wash Buffer AM2
TOTAL REQUIRED

2.025 ml
225 µl2.25 ml

16.2 ml
1.8 ml18 ml

97.2 ml
10.8 ml
108 ml194.4 ml21.6 ml
216 ml1X Antibody
Binding Buffer*

Distilled water
10X Ab Binding Buffer AM3
TOTAL REQUIRED

202.5 µl22.5 µl
225 µl1.62 ml
180 µl
1.8 ml9.72 ml
1.08 ml
10.8 ml19.44 ml
2.16 ml21.6 ml

Developing Solution

TOTAL REQUIRED

112.5 µl

900 µl5.4 ml10.8 ml

Stop Solution

TOTAL REQUIRED

112.5 µl

900 µl5.4 ml10.8 ml* Volumes listed refer to the preparation of buffer for diluting both the primary & secondary
antibodies.

ATF-2 Transcription Factor Assay
Determine the appropriate number of microwell strips required for testing samples, controls andblanks in duplicate. If less than 8 wells in a strip need to be used, cover the unused wells with aportion of the plate sealer while you perform the assay. The content of these wells is stable atroom temperature if kept dry and, therefore, can be used later for a separate assay. Store theunused strips in the aluminum pouch at 4°C. Use the strip holder for the assay.
Prepare the Complete Lysis Buffer, Complete Binding Buffer, 1X Wash Buffer and 1X Antibody
Binding Buffer as described above in the section Buffer Preparation and Recommendations. Multichannel pipettor reservoirs may be used for dispensing the Complete Binding Buffer, Wash Buffer,
Antibody Binding Buffer, Developing Solution and Stop Solution into the wells being used.

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10Step 1: Binding of ATF-2 to its Consensus Sequence1.

Add 40 µl Complete Binding Buffer to each well to be used. If you wish to perform competitive binding experiments, add 40 µl Complete Binding Buffer that contains 20 pmol (2 µl) of
the wild-type or mutated oligonucleotide (see the Buffer Preparation section above for a
description of competitive binding).

2.

Sample wells: Add 10 µl of sample diluted in Complete Lysis Buffer per well. We recommend using 1-10 µg of nuclear extract diluted in Complete Lysis Buffer per well. A protocol
for preparing nuclear extracts is provided on page 13.
Positive control wells: Add 5 µg of the provided HeLa (Anisomycin) nuclear extract in 10 µlof Complete Lysis Buffer per well (2 µl of nuclear extract in 8 µl of Complete Lysis Buffer perwell).
Blank wells: Add 10 µl Complete Lysis Buffer only per well.

Reagents

Blank
wells

Positive Control
no competition

Sampleno competition(Optional)
wild-type(Optional)
mutatedComplete Binding Buffer
Wild-type oligonucleotide
Mutated oligonucleotide
Complete Lysis Buffer
Sample in Complete Lysis Buffer

40 µl
–
–
10 µl
–

40 µl
–
–
–
10 µl

40 µl
–
–
–
10 µl

38 µl
2 µl
–
–
10 µl

38 µl
–l
2 µl
–
10 µl

3.

Use the provided adhesive cover to seal the plate. Incubate for 1 hour at room temperature
with mild agitation (100 rpm on a rocking platform). Keep any unused wells covered during
the remaining steps in order to preserve those wells for future assays. Any unused strips
from the stripwell plate can be placed in the foil bag, sealed with tape and stored at 4°C.

4.

Wash each well 3 times with 200 µl 1X Wash Buffer. For each wash, flick the plate over a sinkto empty the wells, then tap the inverted plate 3 times on absorbent paper towels.

Step 2: Binding of Primary Antibody1.

Add 100 µl of diluted phosphorylated ATF-2 antibody (1:1,000 dilution in 1X Antibody Binding
Buffer) to each well being used, including blank wells.

2.

Cover the plate and incubate for 1 hour at room temperature without agitation.

3.

Wash the wells 3 times with 200 µl 1X Wash Buffer (as described in Step 1, No. 4).

Step 3: Binding of Secondary Antibody1.

Add 100 µl of diluted HRP-conjugated antibody (1:1,000 dilution in 1X Antibody Binding Buffer) to all wells being used.

2.

Cover the plate and incubate for 1 hour at room temperature without agitation.

3.

During this incubation, place the Developing Solution at room temperature.

4.

Wash the wells 4 times with 200 µl 1X Wash Buffer (as described in Step 1, No. 4).

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Step 4: Colorimetric Reaction1.

Add 100 µl Developing Solution to all wells being used.

2.

Incubate 2-10 minutes at room temperature protected from direct light. Please read theCertificate of Analysis supplied with this kit for optimal development time for this specific
kit lot. Monitor the blue color development in the sample wells until it turns medium todark blue. Do not overdevelop.

3.

Add 100 µl Stop Solution. In presence of the acid, the blue color turns yellow.

4.

Read absorbance on a spectrophotometer within 5 minutes at 450 nm with an optionalreference wavelength of 655 nm. Blank the plate reader according to the manufacturer’s
instructions using the blank wells.

References
1.
2.
3.
4.
5.

Yang S.H. et al. (1998) EMBO J. 17: 1740-1749.
Pearson G. et al. (2001) Endocrine Rev. 22(2): 153-183.
Youn H.D. et al. (2000) J. Biol. Chem. 275(29): 22563-22567.
Ramsauer K. et al. (2002) PNAS 99(20): 12859-12864.
Sano Y. et al. (1999) J. Biol. Chem. 274(13): 8949-8957.

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12

Appendix
Section A. Preparation of Nuclear Extract
For your convenience, Active Motif offers a Nuclear Extract Kit (Cat. Nos. 40010 & 40410). This kit
contains buffers optimized for use in TransAM Kits, which serves to reduce inconsistencies in theassay that may arise from using homemade or other buffers. If you prefer to make your own buffers, please refer to the following protocol.
This procedure can be used for a confluent cell layer of 75 cm2 (100-mm dish). The yield is approximately 0.15 mg of nuclear proteins for 9 x 106 cells.
1.

Wash cells with 10 ml ice-cold PBS/PIB. Discard PBS/PIB.

2.

Add 10 ml ice-cold PBS/PIB and scrape the cells off the dish with a cell lifter. Transfer cells
into a pre-chilled 15 ml tube and spin at 300 x g for 5 minutes at 4°C. Do not use Trypsin toremove cells as it may alter transcription factor activation states.

3.

Resuspend the pellet in 1 ml ice-cold HB buffer by gentle pipetting and transfer the cells intoa pre-chilled 1.5 ml tube.

4.

Allow the cells to swell on ice for 15 minutes.

5.

Add 50 µl 10% Nonidet P-40 (0.5 % final) and vortex the tube vigorously for 10 seconds.
Check the cells under a microscope to monitor the cell lysis. The cell membrane should becompletely lysed, while the nuclear membrane remains intact.

6.

Centrifuge the homogenate for 30 seconds at 4°C in a microcentrifuge. Remove the supernatant (cytoplasmic fraction) and, if you wish to save this for other uses, transfer it into a
pre-chilled microcentrifuge tube. (Store the cytoplasmic fraction at –80°C.)

7.

Resuspend the nuclear pellet in 50 µl Complete Lysis Buffer and rock the tube gently on ice
for 30 minutes on a shaking platform.

8.

Centrifuge for 10 minutes at 14,000 x g at 4°C and save the supernatant (nuclear extract).
Aliquot and store at –80°C. Avoid freeze/thaw cycles. Discard the debris pellet.

9.

Determine the protein concentration of the extract by using a Bradford-based assay. It is
recommended to perform a 1:50 dilution of your samples for protein determination. A blanksample consisting of Complete Lysis Buffer diluted 1:50 should be run as control. Standard
controls should also be generated in Complete Lysis Buffer diluted 1:50.

www.activemotif.com

13

Preparation of Buffers for Nuclear Extract
10X PBS					0.1 M phosphate buffer, pH 7.5		

For 250 ml, mix:
3.55 g Na2HPO4 + 0.61 g KH2PO4

1.5 M NaCl				21.9 g

27 mM KCl				

0.5 g

Adjust to 250 ml with distilled water. Prepare a 1X PBS solution by adding 10 ml 10X PBS to 90 ml
distilled water. Sterilize the 1X PBS by filtering through a 0.2 µm filter. The 1X PBS is at pH 7.5. Storethe filter-sterilized 1X PBS solution at 4°C.

PIB (Phosphatase Inhibitor Buffer)		
125 mM NaF				For 10 ml, mix:
52 mg

250 mM β-glycerophosphate		

0.55 g

250 mM p-nitrophenyl phosphate (PNPP)

1.15 g

25 mM NaVO3				

31 mg

Adjust to 10 ml with distilled water. Mix the chemicals by vortexing. Incubate the solution at 50ºC
for 5 minutes. Mix again. Store at –20°C.

PBS/PIB
Prior to use, add 0.5 ml PIB to 10 ml 1X PBS.
HB (Hypotonic Buffer)		

For 50 ml, mix:

20 mM Hepes, pH 7.5		

0.24 g

5 mM NaF			

12 mg

0 µM Na2MoO4			5 µl of a 0.1 M solution

0.1 mM EDTA			

10 µl of a 0.5 M solution

Adjust pH to 7.5 with 1 N NaOH. Adjust volume to 50 ml with distilled water. Sterilize by filtering
through a 0.2 µm filter. Store the filter-sterilized solution at 4°C.

www.activemotif.com

14Section B: Troubleshooting Guide
Problem/question

Possible cause

Recommendation

No signal or weak signal

Omission of key
reagent

Check that all reagents have been added in all wells in thecorrect order

Substrate or conjugate
is no longer active

Test conjugate and substrate for activity by mixing HRP and
Developing Solution together

Enzyme inhibitor
present

Sodium azide will inhibit the peroxidase reaction. Follow ourrecommendations to prepare buffers

Plate reader settings
not optimal

Verify the wavelength and filter settings

Incorrect assay
temperature

Bring Developing Solution and Stop Solution to room temperature before using

Inadequate volume ofDeveloping Solution

Check to make sure that correct volume is delivered by pipette

Measurement time
too long

Stop enzymatic reactions as soon as the positive wells turn
medium-dark blue

Concentration of antibodies is too highIncrease antibody dilutions

Inadequate washing

Ensure all wells are filled with Wash Buffer and follow washingrecommendations

Incomplete washingof wells

Ensure all wells are filled with Wash Buffer and follow washingrecommendations

Well cross-contamination

Follow washing recommendations

Too much nuclear
extract per well

Decrease amount of nuclear extract down to 2.5 µg/well

Concentration of antibodies is too high

Perform antibody titration to determine optimal working
concentration. Start using 1:2,000 for primary antibody and
1:5,000 for the secondary antibody. The sensitivity of the assay
will be decreased

Not enough nuclear
extract per well

Increase amount of nuclear extract. Not to exceed 40 µg/well

Too many freeze/thaw
cycles of extract

Aliquot extract into 5 µl aliquots and store at -80°C to avoid
multiple freeze/thaws

ATF-2 is poorly activated or inactivated innuclear fractions

Perform a time course for ATF-2 activation in the studied cell
line

Nuclear extracts are
not from correct
species

The ATF-2 antibody works in human samples. It has not beentested for cross-reactivity in other species.

Salt concentration
too high in binding
reaction

Reduce amount of extract per well or dialyze extract before
useHigh background in
all wells

Uneven color
development

High background insample wells

No signal or weak signal
in sample wells

www.activemotif.com

15Section C. Related Products
Transcription Factor ELISAs		FormatCatalog No.

TransAM™ AML-1/Runx1		
TransAM™ AML-3/Runx2		
TransAM™ AP-1 Family		TransAM™ AP-1 c-Fos		
TransAM™ AP-1 c-Jun		
TransAM™ AP-1 FosB		
TransAM™ AP-1 JunD		
TransAM™ ATF-2		
TransAM™ c-Myc		
TransAM™ C/EBP a/b		
TransAM™ CREB		
TransAM™ pCREB		
TransAM™ Elk-1		
TransAM™ ER		TransAM™ FKHR (FOXO1)		
TransAM™ GATA Family		TransAM™ GATA-4		
TransAM™ GR		TransAM™ HIF-1		
TransAM™ HNF Family		TransAM™ HNF-1		
TransAM™ IRF-3 (Human)		
TransAM™ IRF-3 (Mouse)		
TransAM™ IRF-7 		
TransAM™ MAPK Family		TransAM™ MEF2		
TransAM™ MyoD		
TransAM™ NF-YA		
TransAM™ NFATc1		
TransAM™ NFkB Family		TransAM™ Flexi NFkB Family		TransAM™ NFkB p50		
TransAM™ Flexi NFkB p50		
TransAM™ NFkB p50 Chemi		
TransAM™ NFkB p52		
TransAM™ NFkB p52 Chemi		
TransAM™ NFkB p65		
TransAM™ Flexi NFkB p65		
TransAM™ NFkB p65 Chemi		
TransAM™ Nrf2		
TransAM™ Oct-4		
TransAM™ p53		
TransAM™ PPARg		
TransAM™ Sp1		
TransAM™ Sp1/Sp3		
TransAM™ STAT Family		TransAM™ STAT3		
TransAM™ T-bet		

1 x 96-well plate1 x 96-well plate2 x 96-well plates
1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate2 x 96-well plates
1 x 96-well plate1 x 96-well plate1 x 96-well plate2 x 96-well plates
1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate2 x 96-well plates
1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate2 x 96-well plates
2 x 96-well plates
1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate2 x 96-well plates
1 x 96-well plate1 x 96-well plate

47396
44496
44296
44096
46096
45096
43496
42396
43396
44196
42096
43096
44396
41396
46396
48296
46496
45496
47096
46296
46196
48396
48496
50196
47296
43196
47196
40396
40296
43296
43298
41096
41098
41097
48196
48197
40096
40098
40097
50296
41196
42496
40196
41296
40496
42296
45196
51396

Function ELISA		FormatCatalog No.

1 x 96-well plate

48005

FunctionELISA IkBa
www.activemotif.com

16

Nuclear Extract		FormatCatalog No.

Nuclear Extract Kit				

100 rxns
400 rxns

40010
40410

Recombinant Proteins		

FormatCatalog No.

Recombinant c-Fos protein		
Recombinant c-Jun protein		
Recombinant c-Myc protein		
Recombinant CREB protein		
Recombinant elF2a protein		
Recombinant NFkB p50 protein		
Recombinant NFkB p65 protein		
Recombinant p53 protein		
Recombinant p300 protein		Purified Sp1 protein		
Recombinant STAT3 protein		

5 µg
5 µg
5 µg
25 µg
25 µg
5 µg
5 µg
5 µg
4 µg
2 µg
10 µg

31115
31116
31117
31107
31108
31101
31102
31103
31124
31137
31140

Cell Extracts		

FormatCatalog No.

HeLa		HeLa (Anisomycin)		

200 µg200 µg

36010
36111

Histone Purification		

FormatCatalog No.

Histone Purification Kit		
Histone Purification Mini Kit		

10 rxns
20 rxns

40025
40026

Co-Immunoprecipitation		

FormatCatalog No.

Nuclear Complex Co-IP Kit		
Universal Magnetic Co-IP Kit		

50 rxns
25 rxns

54001
54002

DNA Methylation		

FormatCatalog No.

MethylDetector™		
MethylCollector™		
MethylCollector™ Ultra		
UnMethylCollector™		

50 rxns
25 rxns
30 rxns
30 rxns

55001
55002
55005
55004

SUMOylation		

FormatCatalog No.

SUMOlink™ SUMO-1 Kit		
SUMOlink™ SUMO-2/3 Kit		

20 rxns
20 rxns

40120
40220

Histone ELISAs		FormatCatalog No.

Histone H3 monomethyl Lys4 ELISA		Histone H3 dimethyl Lys4 ELISA		Histone H3 trimethyl Lys4 ELISA		Histone H3 phospho Ser28 ELISA		

1 x 96 rxns
1 x 96 rxns
1 x 96 rxns
1 x 96 rxns

53101
53102
53103
53100

www.activemotif.com

17

ChIP-validated Antibodies

Application

AP-2 pAb
ChIP, EMSA
ChIP, EMSA, IF, WB
C/EBPa pAb
c-Jun pAb
ChIP, EMSA, IF
DNMT1 mAb
ChIP, IHC, IP, WB
DNMT3A mAb
ChIP, IF, IHC, WB
DNMT3B mAb
ChIP, IF, IP, WB
E2F-1 pAb
ChIP, EMSA
E2F-6 mAb
ChIP, WB
EZH2 pAb
ChIP, IF, IP, WB
HBP-1 mAb
ChIP, IF, WB
HDAC1 mAb (Clone 10E2)
ChIP, IF, IHC, IP, WB
HDAC2 mAb (Clone 3F3)
ChIP, IF, IHC, IP, WB
HDAC3 pAb
ChIP, WBHDAC4 pAb
ChIP, WBHDAC5 pAb
ChIP, WBHDAC6 pAb
ChIP, WB
Histone H2A pAb
ChIP, WB
Histone H2A phospho Ser129 pAb
ChIP, IF, IP, WB
Histone H2B pAb
ChIP, WB
Histone H3 acetyl Lys27 pAb
ChIP, IF, WB
Histone H3 acetyl Lys27 pAb
ChIP, WB
Histone H3 dimethyl Lys4 pAb
ChIP, WB
Histone H3 dimethyl Lys9 pAb
ChIP, IF, WB
Histone H3 trimethyl Lys4 pAb
ChIP, WB
Histone H3 trimethyl Lys9 pAb
ChIP, WB
Histone H3 trimethyl Lys27 mAb
ChIP, WB
Histone H3, C-terminal pAb
ChIP, WB
Histone H4 acetyl Lys5 pAb
ChIP, IF, WB
Histone H4 acetyl Lys12 pAb
ChIP, WB
Histone H4 acetyl Lys16 pAb
ChIP, WB
Histone H4 monomethyl Lys20 pAb
ChIP, IF, WB
Histone H4 trimethyl Lys20 pAb
ChIP, IF, WB
Histone H4 tetra-acetyl pAb
ChIP, WB
HP1 alpha pAb
ChIP, IP, WB
IRF-3 pAb
ChIP, WB
JunB pAb
ChIP, EMSA
JunD pAb
ChIP, EMSAL3MBTL1 pAb
ChIP, IP, WB
Mi-2 beta pAb
ChIP, IF, IP, WB
p53 pAb
ChIP, EMSAPP2A pAb
ChIP, IP, WB
RbAp46/48 pAb
ChIP, WB
RNA pol II mAb
ChIP, ELISA, IF, IP, WB
SNF2h mAb
ChIP, IF, IP, WB
Sp1 pAb
ChIP, WB
TRF2 Goat pAb
ChIP, IP, WB

FormatCatalog No.

17 rxns
100 µg100 µg100 µg100 µg100 µg17 rxns
100 µl200 µl100 µl200 µl200 µl100 µg100 µg100 µg100 µg200 µl200 µl200 µl200 µg200 µl200 µl200 µl200 µl200 µl200 µl200 µl200 µl200 µl200 µl200 µl200 µl50 µl200 µl100 µl17 rxns
100 µl200 µl200 µl17 rxns
200 µl200 µl200 µl200 µl100 µl100 µg

39304
39306
39309
39204
39206
39207
39313
39509
39103
39511
39531
39533
40968
40969
40970
40971
39235
39271
39237
39133
39135
39141
39239
39159
39161
39535
39163
39169
39165
39167
39175
39180
39179
39295
39033
39326
39328
39182
39289
39334
39192
39198
39097
39543
39058
39223

Application Key: ChIP = Chromatin Immunoprecipitation; EMSA = Electrophoretic Mobility Shift Assay;
IF = Immunofluorescence; IHC = Immunohistochemistry; IP = Immunoprecipitation; WB = Western blot;
For an up-to-date list of ChIP-validated antibodies, please visit www.activemotif.com/chipabs

www.activemotif.com

18

ChIP-IT™ Kits		

FormatCatalog No.

ChIP-IT™ Express		ChIP-IT™ Express Enzymatic		
ChIP-IT™ Express HT		
ChIP-IT™ Protein G Magnetic Beads		
ChIP-IT™		
ChIP-IT™ w/o controls		
ChIP-IT™ Shearing Kit		
ChIP-IT™ Enzymatic 		
ChIP-IT™ Enzymatic w/o controls		
Enzymatic Shearing Kit		
Salmon Sperm DNA/Protein G agarose		
ChIP-IT™ Control Kit – Human		
ChIP-IT™ Control Kit – Mouse		ChIP-IT™ Control Kit – Rat		
Ready-to-ChIP HeLa Chromatin		

25 rxns
25 rxns
96 rxns
25 rxns
25 rxns
25 rxns
10 rxns
25 rxns
25 rxns
10 rxns
25 rxns
5 rxns
5 rxns
5 rxns
10 rxns

53008
53009
53018
53014
53001
53004
53002
53006
53007
53005
53003
53010
53011
53012
53015

www.activemotif.com

19Technical Services
If you need assistance at any time, please call Active Motif Technical Service at one of thenumbers listed below.
Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008
USA
Toll Free:877 222 9543
Telephone: 760 431 1263
Fax:
760 431 1351
E-mail:tech_service@activemotif.com
Active Motif Europe
104 Avenue Franklin Roosevelt
B-1330 Rixensart, Belgium
UK Free Phone:		
0800 169 31 47
France Free Phone:
0800 90 99 79
Germany Free Phone: 0800 181 99 10
Telephone: 		+32 (0)2 653 0001
Fax: 		
+32 (0)2 653 0050
E-mail: 		
eurotech@activemotif.com
Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Telephone: +81 3 5225 3638
Fax:
+81 3 5261 8733
E-mail:japantech@activemotif.com
Visit Active Motif on the worldwide web at http://www.activemotif.com
At this site:• Read about who we are, where we are, and what we do• Review data supporting our products and the latest updates• Enter your name into our mailing list to receive our catalog, MotifVations newsletter andnotification of our upcoming products• Share your ideas and results with us• View our job opportunities
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20