TransAM® AP-1 Family

Transcription Factor Assay Kit(version D5)
Catalog Nos. 44296

Copyright 2014 Active Motif, Inc.

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.
© 2017 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
AP-1 Transcription Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Transcription Factor Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
TransAM AP-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Kit Performance and Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Kit Components and Storage
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Protocols
Buffer Preparation and Recommendations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Quick Chart for Preparing Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
AP-1 Transcription Factor Assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Appendix
Section A. Preparation of Nuclear Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Section B. Troubleshooting Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

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Overview
Activator protein-1 (AP-1) is a transcription factor that is activated during the cell cycle to promote
cell survival, differentiation and adaptive responses. Therefore, accurate monitoring of AP-1 activation in cells, tissues and animals is crucial for many biomedical research and drug development
projects. To date, such research projects are tedious and 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 AP-1 Family Kits are designed specifically to detect andquantify AP-1 activation. They contain a 96-well plate to which oligonucleotide containing a TPAresponsive element (TRE) has been immobilized. AP-1 dimers contained in nuclear cell extracts
bind specifically to this oligonucleotide and are detected through use of an antibody directedagainst c-Fos, FosB, Fra-1, c-Jun, JunB or JunD. Addition of a secondary antibody conjugated tohorseradish peroxidase (HRP) provides sensitive colorimetric readout that is easily quantified
by spectrophotometry. The 96-well plate with individual strips of 8 wells is suitable for manual
use or for high-throughput screening applications. TransAM AP-1 Family Kits are available in one
convenient format:

product
TransAM® AP-1 Family

formatcatalog no.

2 x 96 rxns

44296

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

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1Flow Chart of Process

1

5

4

3

2

7

6

8

9

10

11

12

A
B
C
D
E

Cell extractcontaining activatedtranscription factor

F
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.

9

8

7

6

5

4

3

2

1

10

11

12

A
B
C
D
E
F
G
H

Add anti-IgG
HRP conjugate
21

11

01

9

8

7

6

5

4

1 hr.
3

2

1
A
B

C
D
E
F
G

Add developing
and stop solution

H

1 hr.
5

4

3

2

1

7

6

A
B
C
D
E
F
G
H

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2

8

9

10

11

12

Introduction
AP-1 Transcription Factor
The activator protein-1 (AP-1) transcription factors belong to a large family of structurally relatedtranscription factors that includes ATF1-4, c-Fos, c-Jun, c-Myc and C/EBP (see 1-2 for review). The
members of this family, named bZIP, share a dimerization domain with a leucine zipper motif
and a DNA binding domain rich in basic residues (lysines and arginines). AP-1 is composed of a
mixture of heterodimeric complexes of proteins derived from the Fos and Jun families including
c-Fos, FosB, Fra-1, Fra-2, c-Jun, JunB and JunD. Only Jun proteins can form transcriptionally active homodimers within AP-1 members, or heterodimers with CREB/ATF members, to bind the CRE
element (5´-TGACGTCA-3´)1. Primarily, AP-1 dimers bind to DNA on a TPA-response element (TRE)with the 5´-TGA(C/G)TCA-3´ sequence3. Jun-Fos heterodimers form more stable complexes with
TREs. These complexes display stronger transactivating activity than Jun-Jun homodimers4.
Phosphorylation of AP-1 family members by kinases is required for transactivation activity. In
the case of c-Jun, the activation domain is regulated to a large extent by the JNK family of MAP
kinases5. JNK kinases phosphorylate c-Jun at Ser-63, resulting in the binding of c-Jun to the
CBP/p300 family of transcriptional co-activators6. For the Fos proteins, both N- and C-terminal
domains flanking the bZIP domain require phosphorylation for biological activity. The kinases
responsible for activation remain to be determined7-8.
AP-1 expression is induced by multiple stimuli such as serum, growth factors, phorbol esters andoncogenes2. These include peptide growth factors, cytokines of the TGF-β, TNF, and interferon
families, neuronal depolarization and cellular stress. The total increase in AP-1 DNA binding
activity after any stimulus is often much less impressive than would be expected based on
analysis of mRNA expression. This increase is accompanied by a change in the composition ofAP-1 complexes shortly after stimulation in order to modulate transcriptional control9. Upon serum
starvation of human fibroblast cells, Fos and Jun protein production can be induced for up to 4
hours by adding serum. Interestingly, serum starvation lowers basal expression of FosB and c-Fos
but has no significant effect on c-Jun.
AP-1 proteins play a role in the expression of many genes involved in proliferation and cell cycle
progression including neuronal apoptosis, learning process, drug-induced behavioral responses,
bone growth and differentiation, and embryo development. For instance, cell transformation
by oncogenes that function in the growth factor signal transduction pathway, such as ras, rasF
and mek, results in a high increase in AP-1 component protein expression (see 10 for review).
Therefore, AP-1-regulated genes support the invasive process observed during malignancy andmetastasis.

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3

Transcription Factor Assays
To date, three methods are widely used to measure AP-1 activation, either directly or indirectly:1.

AP-1 activation can be determined by Western blot by using antibodies specific for AP-1
proteins. This method is time consuming (up to 2 days once the nuclear cell extracts areprepared), and is not suitable for processing large numbers of samples.

2.

The DNA-binding capacity of AP-1 can be assayed by gel retardation, also called electrophoretic mobility shift assay (EMSA). In this method, nuclear extracts are incubated with aradioactive double-stranded oligonucleotide probe containing the consensus sequence forAP-1 binding. If AP-1 is active in the nuclear extract, it will bind to the probe. To detect phosphorylated AP-1 proteins, a supershift step in the presence of phospho-specific antibodies
is required. Samples are then resolved by electrophoresis on a native polyacrylamide gel,followed by autoradiography. This method is sensitive, but like the previous procedure, it istime consuming (multiple days of gel exposure may be required to achieve sufficient sensitivity) and it cannot be applied to high-throughput screening. Gelshift assays also require
special precautions and equipment for handling radioactivity.

3.

Another method used to assay AP-1 activation is based on reporter genes, typicallyluciferase or β-galactosidase, placed under the control of a promoter containing the TRE sequences. The promoter can be artificial, made of several TRE cis-elements and a TATA box,
or natural, like the collagenase promoter sequence. However, the procedure is limited by
the following issues: (i) reporter gene assays have to be repeated several times to obtainstatistically reliable data; and (ii) reporter gene assays are sensitive to confounding factorsthat may influence the expression level of the reporter gene, and therefore assays have to
be carefully standardized. Reporter gene assays are sensitive and easy to perform with alarge number of samples, but require efficient cell transfection with the reporter plasmid.

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4

TransAM AP-1
AP-1 is a key factor in various and distinct biological functions such as cell proliferation andsurvival, and therefore represents an excellent pharmacological target. However, pharmaceuticalresearch in this field has been limited by the lack of convenient assays suitable for high sample
volume.
To overcome this, Active Motif is introducing a high-throughput assay to quantify AP-1 activation11.
The TransAM Kit combines a fast and user-friendly ELISA format with a sensitive and specificassay for transcription factors. TransAM AP-1 Kits contain a 96-well plate on which has been
immobilized an oligonucleotide that contains a TRE (5´-TGAGTCA-3´). AP-1 contained in nuclear
extract specifically binds to this oligonucleotide. The primary antibodies used in TransAM AP-1
Kits recognize accessible epitopes on c-Fos, FosB, Fra-1, c-Jun, JunB or JunD proteins upon DNA
binding. Addition of a secondary HRP-conjugated antibody provides a sensitive colorimetric readout easily quantified by spectrophotometry. 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 numberof samples can be handled simultaneously, enabling high-throughput automation. This assay is
specific for AP-1 activation and has been shown to be 5-fold more sensitive and 20-fold fasterthan the gel-retardation technique. With the 3.5-hour TransAM procedure, we could detect AP-1
activation using as little as 1.25 µg of nuclear extract from TPA-stimulated cells. A comparable
assay using EMSA required 2.5 µg of nuclear extract and a 3-day autoradiography.
TransAM has many applications including the study of drug potency toward AP-1 related kinase,AP-1 transcriptional activity regulation and protein structure/function studies of AP-1 and its cofactors in areas such as cell differentiation and survival in tumorigenesis, hematopoiesis, learning
process, drug-induced behavioral responses, and many more.

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5Kit Performance and Benefits
TransAM AP-1 Kits are for research use only. Not for use in diagnostic procedures.
Detection limit: < 1.25 µg nuclear extract/well. TransAM AP-1 is 5-fold more sensitive than EMSA.
Range of detection: TransAM provides quantitative results from 0.1 to 20 µg of nuclear extract/well.
2
1.8

AP-1 activation (OD 450 nm)

1.6
1.4
1.2
K-562+TPA
20 pmol wt oligo
20 pmol mut oligo

1
0.8
0.6
0.4
0.2
0

c-Jun

c-Fos

FosB

JunB

JunD

Fra-1

AP-1 family members

AP-1 family profiling for DNA binding activation upon addition of TPA. Nuclear extracts from K-562 cells stimulated with
TPA were assayed for activity of AP-1 family members c-Jun, c-Fos, FosB, JunB, JunD and Fra-1. Each antibody was tested
with 5 µg/well of nuclear extract in the absence or presence of wild-type or mutated consensus binding oligonucleotides
using the TransAM AP-1 Family Kit.

Cross-reactivity:
FosB

c-Fos

Fra-1

c-Jun*

JunB

JunD

Human

+

+

+

+

+

+

Mouse

+

+

-

+

-

+

Rat

+

+

-

nt

-

+

nt = not tested* c-Jun antibody detects c-Jun phosphorylated at S73 and JunD phosphorylated at S100.

Assay time: 3.5 hours. TransAM is 20-fold faster than EMSA.

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6

Kit Components and Storage
Except for the nuclear extract that must be kept at -80ºC, kit components can be stored at -20ºCprior to first use. Then, we recommend storing each component at the temperature indicated in
the table below.
Reagents

Quantity

Storagec-Fos or Fra-1 antibodies

11 µl

4°C for 6 months

JunB or JunD antibodies

11 µl-20°C for 6 months

FosB or Phospho-c-Jun antibodies

22 µl-20°C for 6 months

Anti-rabbit HRP-conjugated antibody

2 x 11 µl

4°C for 6 months

Wild-type oligonucleotide AM7

100 µl (10 pmol/µl)-20°C for 6 months

Mutated oligonucleotide AM7

100 µl (10 pmol/µl)-20°C for 6 months

K-562 (TPA) nuclear extract

40 µl (2.5 µg/µl)-80°C for 6 months

100 µl (1 M)-20°C for 6 months

100 µl

-20°C for 6 months

100 µl (17 µg/µl)-20°C for 6 months

Lysis Buffer AM1

10 ml

4°C for 6 months

Binding Buffer AM2

10 ml

4°C for 6 months

10X Wash Buffer AM2

60 ml

4°C for 6 months

10X Antibody Binding Buffer AM3

2 x 2.2 ml

4°C for 6 months

Developing Solution

2 x 11 ml

4°C for 6 months

Stop Solution

60 ml

4°C for 6 months

96-well AP-1 assay plate2

4°C for 6 months

Plate sealer

2

Dithiothreitol (DTT)
Protease Inhibitor Cocktail
Poly [d(I-C)]

Additional materials required• Multi-channel pipettor• Multi-channel pipettor reservoirs• Rocking platform• Microplate spectrophotometer capable of reading at 450 nm (655 nm as optional reference
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. Our Nuclear Extract Kit can also be purchased separately (Cat. Nos. 40010& 40410). Lysis Buffer AM1 contains phosphatase inhibitors to prevent dephosphorylation of AP-1
during the extract preparation and the assay. The presence of these inhibitors gives a yellow
coloration to Lysis Buffer AM1. Prepare the amount of Complete Lysis Buffer required for the assay by adding 1 µl of 1 M DTT and 10 µl Protease Inhibitor Cocktail per ml of Lysis Buffer AM1 (seethe Quick Chart for Preparing Buffers in this section). Some of the protease inhibitors lose theiractivity after 24 hours once diluted. Therefore, we recommend using the Complete Lysis Buffer
immediately for cell lysis. The remaining amount should be discarded if not used in the same day.
Preparation of Complete Binding Buffer
Prepare the amount of Complete Binding Buffer required for the assay by adding 1 µl of 1 M DTT
and 10 µl of 17 µg/ml poly[d(I-C)] per ml of Binding Buffer AM2 (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 homogenize the buffer by incubating at 50ºC for 2 minutes and mixing prior touse.
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 10X
Antibody Binding Buffer AM3 may form clumps, therefore homogenize the buffer by warming toroom temperature and vortexing for 1 minute prior to use. Dilute the primary antibody with the 1X
Antibody Binding Buffer to 1:500 for the FosB and Phospho-c-Jun antibodies or to 1:1000 for thec-Fos, Fra-1, JunB, JunD and HRP-conjugated secondary antibodies. Depending on 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 notaffect product 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 K-562 (TPA) nuclear extract is provided as a positive control for AP-1 activation. This extract is
optimized to give a strong signal when used at 5 µg/well for c-Fos, FosB, c-Jun, JunB, JunD and
Fra-1. Sufficient extract is supplied for 20 reactions. We recommend aliquoting the extract in 5 µl
fractions and storing at -80ºC. Avoid multiple freeze/thaw cycles of the extract.
Wild-type and mutated consensus oligonucleotides
The wild-type consensus oligonucleotide is provided as a competitor for AP-1 binding in order tomonitor the specificity of the assay. Used at 20 pmol/well, the oligonucleotide will prevent AP-1
binding to the probe immobilized on the plate. Conversely, the mutated consensus oligonucleotide should have no effect on AP-1 binding. Prepare the required amount of wild-type and/or
mutated consensus oligonucleotide by adding 2 µl of appropriate oligonucleotide to 31.8 µl ofComplete Binding Buffer per well being used (see the Quick Chart for Preparing Buffers in thissection). To allow for optimum competition, add the oligonucleotide to the well prior to additionof the nuclear extract.

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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

0.02 µl0.23 µl22.25 µl22.5 µl

0.2 µl
1.8 µl
178.0 µl180.0 µl

1.2 µl10.8 µl
1.068 ml
1.08 ml

2.4 µl21.6 µl2.136 ml
2.16 ml

Complete Binding Buffer

DTT
Poly[d(I-C)]
Binding Buffer AM2
TOTAL REQUIRED

0.03 µl0.34 µl33.4 µl33.8 µl

0.3 µl
2.7 µl267.0 µl270.0 µl

1.8 µl16.2 µl1.602 ml
1.62 ml

3.6 µl32.4 µl
3.2 ml3.24 ml

Complete Binding Buffer
with wild-type ormutated oligonucleotide

Wild-type or mutated oligo
Complete Binding Buffer
TOTAL REQUIRED

2.0 µl31.8 µl33.8 µl

16.0 µl254.0 µl270.0 µl96.0 µl1.524 ml
1.62 ml

N/A
N/A
N/A

1X Wash Buffer

Distilled water
10X Wash Buffer AM2
TOTAL REQUIRED

2.025 ml
225.0 µl2.25 ml

16.2 ml
1.8 ml18.0 ml97.2 ml
10.8 ml
108.0 ml

194.4 ml21.6 ml
216.0 ml

1X Antibody
Binding Buffer*

Distilled water
10X AB Binding Buffer AM3
TOTAL REQUIRED

202.5 µl22.5 µl225.0 µl

1.62 ml
180.0 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 µl900.0 µl

5.4 ml10.8 ml

Stop Solution

TOTAL REQUIRED

112.5 µl900.0 µl

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

AP-1 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 witha portion 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.
Multi-channel 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.

Step 1: Binding of AP-1 to its Consensus Sequence1.

Add 30 µl Complete Binding Buffer to each well to be used. If you wish to perform competitive binding experiments, add 30 µl Complete Binding Buffer that contains 20 pmol (2 µl)of the wild-type or mutated consensus oligonucleotide (see the Buffer Preparation section
above for a description of competitive binding).
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10

2.

Sample wells: Add 20 µl of sample diluted in Complete Lysis Buffer per well. We recommend using 2-20 µg of nuclear extract diluted in Complete Lysis Buffer per well. A protocol
for preparing nuclear extracts can be found on page 12.
Positive control wells: Add 5 µg of the positive control nuclear extract diluted in 20 µl ofComplete Lysis Buffer per well (2 µl of nuclear extract in 18 µl of Complete Lysis Buffer per well).
Please refer to page 9 for recommended positive control information.
Blank wells: Add 20 µl Complete Lysis Buffer only per well.

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).

4.

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

Step 2: Binding of Primary Antibody1.

Add 100 µl of one of the diluted AP-1 antibodies (FosB and Phospho-c-Jun antibodies diluted
to 1:500 in 1X Antibody Binding Buffer, or a 1:1000 dilution in 1X Antibody Binding Buffer forthe c-Fos, Fra-1, JunB or JunD antibodies) to each well being used.

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 (1:1000 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).

Step 4: Colorimetric Reaction1.

Add 100 µl Developing Solution to all wells being used.

2.

Incubate 2-20 minutes at room temperature protected from direct light. Monitor the blue
color development in the sample and positive control wells until it turns medium to dark
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 a reference
wavelength of 655 nm. Blank the plate reader according to the manufacturer’s instructions
using the blank wells.

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11

References
1.
2.
3.

Shaywitz A.J. and Greenberg M.E. (1999) Annu. Rev. Biochem. 68: 821-61.
Wisdom R. (1999) Exp. Cell Res. 253: 180-5.
Angel P., Imagawa M., Chiu R., Stein B., Imbra R.J., Rahmsdorf H.J., Jonat C., Herrlich P. andKarin M. (1987) Cell 49: 729-39.
4. Halazonetis T.D., Georgopoulos K., Greenberg M.E. and Leder P. (1988) Cell 55: 917-24.
5. Karin M. (1995) J. Biol. Chem. 270: 16483-6.
6. Arias J., Alberts A.S., Brindle P., Smeal T., Karin M., Feramisco J. and Montminy M. (1994)
Nature 370: 226-9.
7. Deng T. and Karin M. (1994) Nature 371: 171-5.
8. Skinner M., Qu S., Moore C. and Wisdom R. (1997) Mol. Cell. Biol. 17: 2372-80.
9. Chiu R., Angel P. and Karin M. (1989) Cell 59: 979-86.
10. Ozanne B.W., McGarry L., Spence H.J., Johnston I., Winnie J., Meagher L. and Stapleton G.
(2000) Eur. J. Cancer 36: 1640-8.
11. Renard P., Ernest I., Houbion A., Art M., Le Calvez H., Raes M. and Remacle J. (2001) Nucleic
Acids Res. 29: (4) e21.

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
the assay 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.

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.

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.

9.

Determine the protein concentration of the extract by using a Bradford-based assay.
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12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.
Store the 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

13Section B: Troubleshooting Guide
Problem/question

Possible cause

Recommendation

No signal or weaksignalOmission of key
reagent

Check that all reagents have been added in all wells in
the correct order

Substrate or conjugate is no longer
activeTest conjugate and substrate for activity

Enzyme inhibitor
present

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

Plate reader settings
not optimal

Verify the wavelength and filter settings in the plate
reader

Incorrect assay
temperature

Bring substrate to room temperature

Inadequate volumeof Developing
Solution

Check to make sure that correct volume is delivered by
pipetteDeveloping time
too long

Stop enzymatic reaction 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
washing recommendations

Incomplete washingof wells

Ensure all wells are filled with Wash Buffer and follow
washing recommendations

Well cross-contamination

Follow washing recommendations

Too much nuclear
extract per well

Decrease amount of nuclear extract down to 1-2 µg/well

Concentration of
antibodies is too
high

Perform antibody titration to determine optimal working
concentration. Start using 1:1000 for primary antibody
and 1:5000 for the secondary antibody. The sensitivityof the assay will be decreased

Not enough nuclear
extract per well

Increase amount of nuclear extract to 50 µg/wellAP-1 is poorly activated or inactivatedin nuclear fractions

Perform a time course for AP-1 activation in the studied
cell line

High background in
all wells

Uneven color
development

High background insample wells

No signal or weaksignalin sample wells

Nuclear extracts
are not from correct
specieswww.activemotif.com

14

Technical Services
If you need assistance at any time, please call or send an e-mail to Active Motif Technical Service
at one of the locations listed below.

Active Motif North America
Toll free:Direct:Fax:
E-mail:Active Motif Europe
UK Free Phone:France Free Phone:
Germany Free Phone:Direct:Fax:
E-mail:877.222.9543
760.431.1263
760.431.1351
tech_service@activemotif.com
0800/169 31 47
0800/90 99 79
0800/181 99 10+32 (0)2 653 0001
+32 (0)2 653 0050
eurotech@activemotif.com

Active Motif Japan
Direct: +81 (0)3 5225 3638
Fax: +81 (0)3 5261 8733
E-mail: japantech@activemotif.com
Active Motif China
Direct: (86)-21-20926090
Cell Phone: 18521362870
E-mail: techchina@activemotif.com

Visit Active Motif online at activemotif.com

www.activemotif.com

15