TransAM® MAPK Family

Transcription Factor Assay Kits
(version B2)
Catalog No. 47296

Revision

Date

Description of Change

B2

August, 2023

c-Myc antibody has changed to new monoclonal
antibody. However, dilution used in protocol andspecies reactivity are the same as previous.

TABLE OF CONTENTS

Page

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Flow Chart of Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Introduction
MAPK Regulated Transcription Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Transcription Factor Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
TransAM MAPK Family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Kit Performance and Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Protocols
Buffer Preparation and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Quick Chart for Preparing Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
MAPK Transcription Factor Assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Preparation of Nuclear Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Appendix
Section A. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

www.activemotif.com

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 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 MAPK Family Kits are designed specifically for the study of
the MAPK regulated transcription factors ATF-2, c-Jun, c-Myc, MEF2 and STAT1. The kit contains
a 96-well plate to which a mixture of oligonucleotides containing the consensus-binding sites for
ATF-2, c-Jun, c-Myc, MEF2 and STAT1 have been immobilized. The activated form of the protein
contained in nuclear extracts specifically binds to this oligonucleotide. By using an antibody thatis directed against phosphorylated ATF-2, phosphorylated c-Jun, c-Myc, MEF2 or STAT1α, thecomplex bound to the oligonucleotide is detected. Addition of a secondary antibody conjugated tohorseradish peroxidase (HRP) provides a sensitive colorimetric readout that is easily quantified byspectrophotometry. The 96-well plate with individual strips of 8 wells is suitable for manual use or
high-throughput screening applications.
product
TransAM MAPK Family

formatcatalog no.

2 x 96-well plates

47296

See Active Motif products related to MAPK signaling pathways in Appendix, Section B.

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

www.activemotif.com

1Flow Chart of Process

www.activemotif.com

2

Introduction
MAPK Regulated Transcription Factors
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
and cytoplasmic 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 and differentiation,
glycolysis and apoptosis. Deregulation of c-Myc has been implicated in the origin of diverse
human cancers. 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 theregulation of the activity of the c-Fos promoter in response to extracellular stimuli.2 MEF2, amember of the MADS box 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 activatedby 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

www.activemotif.com

3

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

Expression can be measured by Western blot, using antibodies raised against the transcription factor of interest. This method is time consuming (up to 2 days once the cell extracts
are prepared), and is not suitable for processing large numbers of samples.

2.

The DNA-binding capacity of MAPK regulated transcription factors can be assayed by gel
retardation, also called electrophoretic mobility shift assay (EMSA). In this method, nuclear
extracts are incubated with a radioactive double-stranded oligonucleotide probe containing the consensus sequence for binding. If the transcription factor is active in the nuclear
extract, it will bind to the probe. Samples are then resolved by electrophoresis on a nativepolyacrylamide gel, followed by autoradiography. This method is sensitive, but like theprevious procedure, it is time 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 transcription factor activation is based on reporter genes,
typically luciferase or β-galactosidase, placed under the control of a promoter containing
the consensus sequence. This promoter can be artificial, made of several 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 andeasy to perform with a large number of samples but requires efficient cell transfection withthe reporter plasmid.

TransAM MAPKTranscription 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 factors ATF-2, c-Jun, c-Myc, MEF2 and STAT1α. The TransAM Kit combines afast and user-friendly ELISA format with a sensitive and specific assay for transcription factors.
TransAM MAPK Family Kits contain a 96-well plate on which has been immobilized a mixtureof oligonucleotides that contains the consensus binding site for each transcription factor. The
active form of each transcription factor contained in the nuclear extract specifically binds to thisoligonucleotide mixture. The primary antibodies used to detect the MAPK regulated transcription factors will recognize an epitope on phosphorylated ATF-2, phosphorylated c-Jun, c-Myc,
MEF2 and STAT1α that is accessible only when they are activated and bound to their target DNA.
Addition of a secondary HRP-conjugated antibody provides a sensitive colorimetric readout
easily quantified by spectrophotometry. Once the nuclear extracts are prepared, this assay iscompleted in less than 3.5 hours. As this assay is performed in 96-well plates, a large number
www.activemotif.com

4

of samples can be handled simultaneously, enabling high-throughput automation. This assay is
specific for ATF-2, c-Jun, c-Myc, MEF2 and STAT1α activation and has been shown to be 10-foldmore 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 0.5 µg of nuclear extract. A comparable
assay using EMSA required 5 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.

Kit Performance and Benefits
The TransAM MAPK Family Kit is for research use only. Not for use in diagnostic procedures.
Detection limit: < 0.5 µg nuclear extract/well. The TransAM MAPK Family Kit is up to 10-foldmore sensitive than EMSA.
Range of detection: TransAM provides quantitative results from 0.5 to 5 µg of nuclear extract/well for c-Myc, MEF2 and STAT1 and from 1 to 10 µg nuclear extract/well for ATF-2 and c-Jun.
Cross-reactivity: TransAM MAPK Family detects phosphorylated ATF-2, MEF2 and STAT1α fromhuman origin and c-Myc from mouse and human origin. TransAM MAPK Family also detects
mouse and human c-Jun phosphorylated at Ser-73 and JunD phosphorylated at Ser-100, as thissite is conserved between c-Jun and JunD.

MAPK activation (OD450 nm)

Assay time: 3.5 hours. TransAM is 20-fold faster than EMSA.
1.2

Extract
Extract with wild-typecompetitor oligos

1
0.8

Extract with mutant
competitor oligos

0.6
0.4
0.2
0

ATF-2

c-Myc
c-Jun
STAT1
STAT family members

MEF2

Monitoring MAPK regulated transcription factor activity with the TransAM MAPK Family Kit: Nuclear
extracts from HeLa (Anisomycin) for ATF-2, Jurkat (1 day growth) for c-Myc, K-562 (TPA) for c-Jun, U-937 (TPA +IFNγ) for STAT1 and C2C12 for MEF2 were assayed for activity using the TransAM MAPK Family Kit. This data isprovided for demonstration only.

www.activemotif.com

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
2 plates

Storage / Stability

Phosphorylated ATF-2 antibody

11 µl

4°C for 6 months

Phosphorylated c-Jun antibody

22 µl-20°C for 6 months

c-Myc antibody

11 µl-20°C for 6 months

MEF2 antibody

11 µl

4°C for 6 months

STAT1α antibody

11 µl-20°C for 6 months

HRP-rabbit conjugated IgG

2 x 11 µl(0.2 µg/µl)

4°C for 6 months

HRP-mouse conjugated IgG

11 µl / 55 µl(0.2 µg/µl)

4°C for 6 months

AP-1 wild-type oligonucleotide AM7

100 µl(10 pmol/µl)-20°C for 6 months

AP-1 mutated oligonucleotide AM7

100 µl(10 pmol/µl)-20°C for 6 months

ATF-2 wild-type oligonucleotide AM2

100 µl(10 pmol/µl)-20°C for 6 months

ATF-2 mutated oligonucleotide AM2

100 µl(10 pmol/µl)-20°C for 6 months

c-Myc wild-type oligonucleotide AM3

100 µl(10 pmol/µl)-20°C for 6 months

c-Myc mutated oligonucleotide AM3

100 µl(10 pmol/µl)-20°C for 6 months

MEF2 wild-type oligonucleotide AM5

100 µl(10 pmol/µl)-20°C for 6 months

MEF2 mutated oligonucleotide AM5

100 µl(10 pmol/µl)-20°C for 6 months

STAT wild-type oligonucleotide AM6

100 µl(10 pmol/µl)-20°C for 6 months

STAT mutated oligonucleotide AM6

100 µl(10 pmol/µl)-20°C for 6 months

K-562 nuclear extract (TPA stimulated)

40 µl(2.5 µg/µl)-80°C for 6 months

Dithiothreitol (DTT)

100 µl
(1 M)-20°C for 6 months

Protease Inhibitor Cocktail

100 µl

-20°C for 6 months

Reagents

www.activemotif.com

6

Lysis Buffer AM1

10 ml

4°C for 6 months

Binding Buffer AM6
months

10 ml

4°C for 6

10X Wash Buffer AM2
months

60 ml

4°C for 6

10X Antibody Binding Buffer AM3
months2 x 2.2 ml

4°C for 6

Developing Solution
months2 x 11 ml

4°C for 6

Stop Solution
months

60 ml

4°C for 6

96-well assay platemonths2

4°C for 6

Plate sealer2

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)
For Nuclear Extract preparation• Hypotonic Buffer• Phosphatase Inhibitor Buffer• 10X PBS
• Detergent (NP-40)

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). Prepare the amount of Complete Lysis Buffer required for the assay by adding 1 µl of
1 M DTT and 10 µl of Protease Inhibitor Cocktail per ml of Lysis Buffer AM1 (see the Quick Chartfor Preparing Buffers in this section). Some of the protease inhibitors lose their activity after 24hours once diluted. Therefore, we recommend using the Complete Lysis Buffer immediately forcell 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 2 µl of 1 M DTT
per ml of Binding Buffer AM6 (see the Quick Chart for Preparing Buffers in this section). Afteruse, 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 vortexing for 2 minutes 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 homogenize the buffer by warming to room temperature and vortexing for 1 minute prior to use. Dilute the phosphorylated c-Jun
antibody to 1:500 and all other primary and secondary antibodies to 1:1000 with the 1X Antibody
Binding Buffer. Depending on the particular assay, the signal:noise ratio may be optimized byusing 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.

www.activemotif.com

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 atroom temperature for at least 1 hour. Transfer the amount of Developing Solution required forthe 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
K-562 (TPA stimulated) nuclear extract is provided as a positive control to ensure the kit
reagents are functional. The K-562 (TPA stimulated) extract is optimized to give a strong signal
for bothc-Jun and ATF-2 when used at 5 µg/well. Sufficient extract is supplied for 20 reactions. We
recommend aliquoting the extract in 5 µl fractions and storing at -80°C to avoid multiple freeze/
thaw cycles of the extract.
In addition, positive control nuclear extract for c-Myc, MEF2 and STAT1 can be purchased separately. Jurkat nuclear extract (1 day growth) is recommended as a control for c-Myc. C2C12(Undifferentiated) nuclear extract is suitable for MEF2 and COS-7 nuclear extract (IFNγ treated) is
suitable for STAT1 (see Appendix, Section B. Related Products).
Wild-type and mutated consensus oligonucleotides
The wild-type consensus oligonucleotides are provided as competitors for binding in order tomonitor the specificity of the assay. Used at 20 pmol/well, the oligonucleotide will prevent binding to the probe immobilized on the plate. Conversely, the mutated consensus oligonucleotide
should have no effect on binding. Prepare the required amount of wild-type and/or mutatedconsensus oligonucleotide by adding 2 µl of appropriate oligonucleotide to 43 µl of Complete
Binding Buffer per well being used (see the Quick Chart for Preparing Buffers in this section). To
allow for optimum competition, add the oligonucleotide to the well prior to addition of the cell
extract.

www.activemotif.com

9Quick Chart for Preparing Buffers
For 1
well

For 1 strip(8 wells)

For 6 strips(48 wells)

For 12 strips(96 wells)

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

Reagents to prepare

Components

Complete Lysis Buffer* Volumes listed refer to the preparation of buffer for diluting both the primary & secondary antibodies.

MAPK 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.

www.activemotif.com

10Step 1: Binding of MAPK-regulated transcription factors to their 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 consensus 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 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 10 µl ofComplete Lysis Buffer per well (2 µl of extract in 8 µl of Complete Lysis Buffer per well).
Please refer to page 9 for specific positive control recommendations.
Blank wells: Add 10 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 Washing 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 antibodies (1:500 dilution for phosphorylated c-Jun and1:1000 dilution for all other antibodies in 1X Antibody Binding Buffer) to each well.

2.

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

3.

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

Step 3: Binding of secondary antibody1.

Add 100 µl diluted HRP-conjugated antibody (1:1000 dilution in 1X Antibody Binding Buffer)to all wells being used. Use the mouse HRP with the phosphorylated ATF-2 antibody; allother antibodies require the rabbit HRP.

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 Washing Buffer (as described in Step 1, No. 4).

Step 4: Colorimetric reaction1.

Add 100 µl room-temperature Developing Solution to all wells being used.

2.

Incubate 2-10 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.

www.activemotif.com

11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 isapproximately 0.5 mg of nuclear proteins for 107 cells.
1.

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

2.

Add 10 ml of ice-cold PBS/PIB and scrape the cells off the dish with a cell lifter. Transfer thecells 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 of ice-cold HB buffer by gentle pipetting and transfer the cells
into a 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 mix by gentle pipetting.

6.

Centrifuge the homogenate for 30 seconds at 4°C in a microcentrifuge.

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 cell
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.

10X PBS
0.1 M phosphate buffer, pH 7.5
1.5 M NaCl
27 mM KCl

For 250 ml, mix:
3.55 g Na2HPO4 + 0.61 g KH2PO4
21.9 g
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
250 mM β-glycerophosphate
250 mM para-nitrophenyl phosphate (PNPP)
25 mM NaVO3

For 10 ml, mix
52 mg
0.55 g1.15 g
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.

www.activemotif.com

12PBS/PIB
Prior to use, add 0.5 ml of PIB to 10 ml of 1X PBS.
HB (Hypotonic Buffer)
20 mM Hepes, pH 7.5
5 mM NaF
10 µM Na2MoO4
0.1 mM EDTA

For 50 ml, mix
0.24 g
12 mg
5 µl of a 0.1 M solution
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.

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.

www.activemotif.com

13

Appendix
Section A. Troubleshooting Guide
PROBLEMPOSSIBLE CAUSE

RECOMMENDATION

No signal or weak signal
in all wells

Omission of key reagent

Check that all reagents have been addedin the correct order

Substrate or conjugate is nolonger active

Test 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 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
too high

Increase antibody dilutions

Inadequate washing

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

Incomplete washing of 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
too high

Perform antibody titration to determine
optimal working concentration. Start using
1:2000 for primary antibody and 1:5000 for thesecondary antibody. The sensitivity of theassay will be decreased

Not enough nuclear extract per well

Increase amount of nuclear extract not toexceed 40 µg/well

The factor is poorly activated orinactivated

Perform a time course for activationin the studied cell line

Extracts are not fromcorrect species

Refer to cross-reactivity information on page 5High background in all wells

Uneven color development

High background in sample wells

No signal or weak signal insample wells

www.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

Notes