TransAM®

AP-1 c-Fos/FosB/c-Jun/JunD
Transcription Factor Assay Kits
(version D8)
Catalog Nos. 44096 & 44596 (c-Fos)
Catalog Nos. 45096 & 45596 (FosB)
Catalog Nos. 46096 & 46596 (c-Jun)
Catalog Nos. 43496 & 43996 (JunD)

Copyright 2017 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
TransAM AP-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Kit Performance and Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Kit Components and Storage
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Protocols
Buffer Preparation and Recommendations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Quick Chart for Preparing Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
AP-1 Transcription Factor Assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Appendix
Section A. Preparation of Nuclear Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section B. Troubleshooting Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

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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 witha sensitive, specific assay. TransAM AP-1 Kits are designed specifically to detect and quantify AP-1
activation. They contain a 96-well plate to which oligonucleotide containing a TPA-responsive
element (TRE) has been immobilized. AP-1 dimers contained in nuclear extracts bind specificallyto this oligonucleotide and are detected through use of an antibody directed against c-Fos, FosB,
c-Jun or JunD. Addition of a secondary antibody conjugated to horseradish peroxidase (HRP) provides sensitive colorimetric readout that is easily quantified by spectrophotometry. The 96-wellplate with individual strips of 8 wells is suitable for manual use or for high-throughput screening
applications. TransAM AP-1 Kits are available in two sizes:
product

formatcatalog no.

TransAM® AP-1 c-Fos

1 x 96-well plate5 x 96 well plates

44096
44596

TransAM® AP-1 FosB

1 x 96-well plate5 x 96 well plates

45096
45596

TransAM® AP-1 c-Jun

1 x 96-well plate5 x 96 well plates

46096
46596

TransAM® AP-1 JunD

1 x 96-well plate5 x 96 well plates

43496
43996

The procedure outlined in this manual can be used for either the TransAM AP-1 c-Fos, TransAM
AP-1 FosB, TransAM AP-1 c-Jun or TransAM AP-1 JunD Kits.
Active Motif also offers the TransAM AP-1 Family Kit for the study of AP-1 c-Fos, FosB, Fra-1, c-Jun,
JunB and JunD. See this and other Active Motif products related to AP-1 in Appendix, Section C.

* 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 11 for review).
Therefore, AP-1-regulated genes support the invasive process observed during malignancy andmetastasis.
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.
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3

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.

TransAM AP-1
AP-1 is a key factor in various and distinct biological functions such as cell proliferation and survival,
and therefore represents an excellent pharmacological target. However, pharmaceutical research inthis 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 activation12.
The TransAM Kit combines a fast and user-friendly ELISA format with a sensitive and specific assay
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 specificallybinds to this oligonucleotide. The primary antibodies used in TransAM AP-1 Kits recognize accessible
epitopes on c-Fos, FosB, c-Jun or JunD proteins upon DNA binding. Addition of a secondary HRPconjugated 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 thisassay is performed in 96-well plates, a large number of 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 faster than 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
PMA-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 inareas such as cell differentiation and survival in tumorigenesis, hematopoiesis, learning process,drug-induced behavioral responses, and many more.
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4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.

AP-1 activation (OD450nm )2.5

y = 0.204x + 0.224r 2 = 0.979

2

1.5

Unstim.
PMA/I -stim.

1

0.5

0

0

2.5

5

7.5

10

12.5

Extract (g/well)
Monitoring AP-1 activation with the TransAM AP-1 c-Fos Kit. Different amounts of nuclear extracts from unstimulated(opened squares) and PMA/Ionomycin-stimulated (filled circles) WI-38 cells are tested for AP-1 activation by using theTransAM AP-1 c-Fos Kit. These curves are provided for demonstration only.

Cross-reactivity:
FosB

c-Fos

c-Jun*

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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5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.
Quantity
1 plate / 5 plates

Storagec-Fos antibody

11 µl / 55 µl

4°C for 6 months

JunD antibody

11 µl / 55 µl-20°C for 6 months

FosB or Phospho-c-Jun antibody

22 µl / 110 µl-20°C for 6 months

Anti-rabbit HRP-conjugated antibody

11 µl / 55 µl

4°C for 6 months

Wild-type oligonucleotide AM7

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

Mutated oligonucleotide AM7

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

K-562 (TPA) nuclear extract

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

Dithiothreitol (DTT)

100 µl / 500 µl-20°C for 6 months

Protease Inhibitor Cocktail

100 µl / 500 µl-20°C for 6 months

Poly [d(I-C)]

100 µl / 500 µl(17 µg/µl)-20°C for 6 months

Lysis Buffer AM1

10 ml / 50 ml

4°C for 6 months

Binding Buffer AM2

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 AP-1 assay plate

1/5

4°C for 6 months

Plate sealer

1/5

Reagents(1 M)

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

6

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 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 or Phospho-c-Jun antibodies or to 1:1000 for
the c-Fos, 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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7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 stimulated) nuclear extract is provided as a positive control for AP-1 activation.
Nuclear extract was made from K-562 cells that had been stimulated with 50 ng/ml TPA for 2
hours at 37ºC. Sufficient extract is supplied for 20 reactions. This extract is optimized to give astrong signal when used at 5 µg/well. We recommend aliquoting the extract in 5 µl fractions andstoring at -80ºC. Avoid multiple freeze/thaw cycles of the extract. Various cell extracts are available 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 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.
OPTIONAL – Preparation of standard curve
For those who wish to quantify the amount of AP-1 in their samples, Active Motif offers recombinantc-Fos and c-Jun for use as protein standards (see Appendix, Section C. Related Products).
1.

Begin with a 100 ng/µl working stock of recombinant protein (use the TransAM Complete
Lysis Buffer to dilute the protein). Set up a standard curve in duplicate using the following
concentrations: 16, 8, 4, 2, 1, 0.5, 0.25 and 0 ng/µl. Note: The preceding range is provided as
guidance, a broader range of values may be used.

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8

2.

Make up a 16 ng/µl solution by adding 16 µl of the 100 ng/µl working stock to 84 µl ofComplete Lysis Buffer. Next, pipette 50 µl of Complete Lysis Buffer into the 6 remaining
tubes. Use the 16 ng/µl solution to prepare a dilution series as indicated below. Be sure tomix each tube thoroughly before each transfer. The 16 ng/µl standard serves as the highstandard, while Complete Lysis Buffer alone serves as the 0.0 ng/µl.
50 µl

16 ng/µl
3.

50 µl

8 ng/µl50 µl

4 ng/µl50 µl

2 ng/µl50 µl

1 ng/µl50 µl

0.5 ng/µl

0.25 ng/µl

20 µl from each tube will be aliquoted to the wells in Step 1, No. 2 of the protocol and will correspond to the following quantities of AP-1: 320, 160, 80, 40, 20, 10, 5 and 0.0 ng/well.

Quick Chart for Preparing Buffers
Reagents to prepare

Components

Complete Lysis Buffer

DTT
Protease inhibitor cocktail
Lysis Buffer AM1

Complete Binding Buffer

For 1
well

For 1 strip(8 wells)

For 6 strips(48 wells)

For 12 strips(96 wells)

0.02 µl0.23 µl22.25 µl

0.2 µl
1.8 µl
178.0 µl

1.2 µl10.8 µl
1.068 ml

2.4 µl21.6 µl2.136 ml

TOTAL REQUIRED

22.5 µl180.0 µl1.08 ml

2.16 ml

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

0.03 µl0.34 µl33.4 µl

0.3 µl
2.7 µl267.0 µl

1.8 µl16.2 µl1.602 ml

3.6 µl32.4 µl
3.2 mlTOTAL REQUIRED

33.8 µl270.0 µl

1.62 ml3.24 ml

Complete Binding Buffer
with wild-type ormutated oligonucleotide

Wld-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

2.025 ml
225.0 µl

16.2 ml
1.8 ml97.2 ml
10.8 ml

194.4 ml21.6 ml

TOTAL REQUIRED

2.25 ml18.0 ml

108.0 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 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.

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9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).

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 13.
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 8 for recommended positive control information.
Blank wells: Add 20 µl Complete Lysis Buffer only per well.
OPTIONAL – Protein standard wells: Add 20 µl of the appropriate protein standard dilutedin Complete Lysis Buffer to each well being used (see page 8, Preparation of standard
curve).

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 diluted AP-1 antibody (FosB or Phospho-c-Jun antibodies diluted to 1:500 in 1X
Antibody Binding Buffer, or a 1:1000 dilution in 1X Antibody Binding Buffer for the c-Fos or
JunD antibodies) to all wells 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).

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10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. Please read theCertificate of Analysis supplied with this kit for the optimal development time for this specific kit lot, which varies from lot to lot. 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.

OPTIONAL – Calculation of results using the standard curve
If you have generated a standard curve using Active Motif’s recombinant c-Fos or c-Jun protein,
average the duplicate readings for each standard, control, and sample and subtract the opticaldensity (OD) obtained from the zero standard.
Plot the OD for the standards against the quantity (ng/well) of the standards and draw the best fit
curve. The data can be linearized using log/log paper and regression analysis may also be applied.
To quantify the amount of AP-1 in the samples, find the absorbance value for the samples on the
y-axis and extend a horizontal line to the standard curve. At the intersection point extend a vertical line to the x-axis and read the corresponding standard value. Note: If the samples have been
diluted, the value read from the standard curve must be multiplied by the dilution factor.
See the example curve on the next page.

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11phospho-c-Jun activation (OD450)

Example curve:
The following standard curve is provided for demonstration only. A standard curve should bemade every time an experiment is performed.
c-Jun Recombinant Protein
Signal range 40-320 ng/well

1.25

y = 0.003x + 0.044 r2 = 0.989

1

0.75
0.5

0.25
0

0

100

200

300

400

c-Jun Recombinant Protein (ng)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. Lallemand D., Spyrou G., Yaniv M. and Pfarr C.M. (1997) Oncogene 14: 819-30.
11. Ozanne B.W., McGarry L., Spence H.J., Johnston I., Winnie J., Meagher L. and Stapleton G.
(2000) Eur. J. Cancer 36: 1640-8.
12. Renard P., Ernest I., Houbion A., Art M., Le Calvez H., Raes M. and Remacle J. (2001) Nucleic
Acids Res. 29: (4) e21.

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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
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.

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.

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13

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.

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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

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 in the plate reader

Incorrect assay
temperature

Bring substrate to room temperature

Inadequate volume ofDeveloping Solution

Check to make sure that correct volume is delivered by pipette

Developing 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 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 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 sensitivity of the assay will bedecreased

Not enough nuclear
extract per well

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

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

Nuclear extracts
are not from correct
species

Confirm species cross-reactivity on page 5.

High background in
all wells

Uneven color
development

High background insample wells

No signal or weak signal
in sample wells

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15

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

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16