TransAM™

CREB / pCREB
Transcription Factor Assay Kits
(version D3)
Catalog Nos. 42096 & 42596 (CREB)
Catalog Nos. 43096 & 43596 (pCREB)
Copyright 2010 Active Motif, Inc.

Revision History
Revision

Date

Description of Change

D2

Dec 2018

Updated Related Products available

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.
© 2010 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
CREB Transcription Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Transcription Factor Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
TransAM CREB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Kit Performance and Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Protocols
Buffer Preparation and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Quick Chart for Preparing Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
CREB Transcription Factor Assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
Appendix
Section A: Preparation of Nuclear Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Section B: Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

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Overview
Cyclic AMP Response Element-Binding protein (CREB) is a transcription factor that is activatedduring changes in the cell environment to promote cell survival, differentiation and adaptive responses. Therefore, accurate monitoring of CREB 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 CREB and pCREB Kits are designed specifically to detectand quantify CREB activation. They contain a 96-well plate to which oligonucleotide containing acAMP-responsive element (CRE) has been immobilized. The CREB contained in nuclear extracts
specifically binds to this oligonucleotide and is detected through use of an antibody directedagainst either total CREB or phosphorylated CREB (pCREB). Addition of a secondary antibody
conjugated to horseradish peroxidase (HRP) provides a 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 CREB and pCREB Kits areavailable in two sizes:

product

formatcatalog no.

TransAM CREB

1 x 96-well plate5 x 96-well plates

42096
42596

TransAM pCREB

1 x 96-well plate5 x 96-well plates

43096
43596

See Active Motif products related to the CREB transcription factor in Appendix, Section C.

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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
F

Cell extractcontaining activatedtranscription factor

G
H

Oligonucleotide
coated plate

Add cell
extract
21

11

01

9

8

7

6

5

4

3

2

1
A
B

C
D
E
F
G
H

Add primary
antibody

1 hr.
2

1

9

8

7

6

5

4

3

11

10

12

A
B
C
D
E
F
G
H

Add anti-IgG
HRP conjugate
21

11

01

9

8

7

6

5

1 hr.

4

3

2

1
A
B

C
D
E
F
G

Add developing
and stop solution

H

1 hr.
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
CREB Transcription Factor
Cyclic AMP Response Element-Binding protein (CREB) is a member of a large family of structurally related transcription factors that includes AFT1-4, c-Fos, c-Jun, c-Myc and C/EBP (see 1 forreview). 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). CREB proteinsspecifically recognize the cAMP-responsive element (CRE) promoter site (5´-TGACGTCA-3´)2.
Alternative splicing of the CREB gene yields several forms of CREB protein. The three mostabundant forms are CREBα (341 aa), also called CREB1, CREBΔ (327 aa) and CREBβ (301 and 387aa), which are present in human, rat and mouse tissues3, 4. Two other gene products highly homologous to CREB-1 have been characterized: activating transcription factor 1 (ATF-1)5 and cAMP
response element modulator (CREM)6.
The CREB family members bind to the CRE promoter site as homo- and heterodimers7. The ratio
between these homo- and heterodimers, which depends on the cell type, regulates the CREB
transcriptional activity as the homodimers have a longer half-life than CREB/ATF-1 heterodimers8.
Whereas ATF2-4 are able to heterodimerize with other bZIP factors, such as c-Jun and c-Fos5, 9,
the CREB-1, CREM and ATF-1 proteins only heterodimerize between each other. Therefore, CREB1, CREM and ATF-1 members may represent a distinctive group with the CREB family.
The CREB proteins activate transcription of target genes in response to a diverse array of stimuli,
such as peptide hormones, growth factors and neuronal activity. Activation of CREB is mediatedby a variety of protein kinases including protein kinase A (PKA), mitogen-activated protein kinases (MAPKs) and Ca2+/calmodulin-dependent protein kinases (CaMKs) that phosphorylate CREB at
the Ser-133 residue10. Phosphorylation of Ser 133 is required for CREB-mediated transcription but
not for dimerization and DNA-binding activity. Phosphorylation does, however, increase CREB’s
affinity for its promoter site (see 1 for review). Upon cell stimulation with forskolin, an activator of
adenylyl cyclase11, the kinases are activated by cAMP production and translocate to the nucleus
where they phosphorylate CREB at Ser 13312. Therefore, CREB is almost exclusively nuclear inboth unstimulated and stimulated cells. A cofactor, CREB-binding protein (CPB), specifically binds
to phosphorylated CREB to enhance transcriptional activity13.
CREB is critical for a variety of cellular processes such as cell proliferation, differentiation andadaptive responses. CREB is implicated in the learning and memorization processes14 andcontributes to neuronal adaptation to drugs of abuse15. CREB activity is also important for hormonal control of metabolic pathways, such as gluconeogenesis, by the hormones glucagon andinsulin16.

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

CREB phosphorylation can be determined by Western Blot by using antibodies specificfor Ser-133 phosphorylated CREB. This method is time consuming (up to 2 days once thenuclear extracts are prepared), and is not suitable for processing large numbers of samples.

2.

The DNA-binding capacity of CREB 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
for CREB binding. If CREB is active in the nuclear extract, it will bind to the probe. To detect
phosphorylated CREB, a supershift step in the presence of phospho-specific antibodies isrequired. 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. Gel-shift assays also require
special precautions and equipment for handling radioactivity.

3.

Another method used to assay CREB activation is based on reporter genes, typicallyluciferase or β-galactosidase, placed under the control of a promoter containing the CRE
sequence. The promoter can be artificial, made of several CRE cis-elements and a TATA box,
or natural, like the somatostatin 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 CREB
CREB is a key factor in various immune and neuronal processes, and therefore represents an
excellent pharmacological target. However, pharmaceutical research in this field has been limitedby the lack of convenient assays suitable for high sample volume.
To overcome this, Active Motif is introducing a high-throughput assay to quantify CREB activation17. The TransAM Kit combines a fast and user-friendly ELISA format with a sensitive and specific assay for transcription factors. TransAM CREB and pCREB Kits contain a 96-well plate on which
has been immobilized an oligonucleotide that contains a CRE (5´-TGACGTCA-3´). CREB containedin nuclear extract specifically binds to this oligonucleotide. The primary antibody used in TransAM
CREB Kits recognizes detects endogenous levels of total CREB (independent of the phosphorylation state of CREB). In contrast, the primary antibody used in TransAM pCREB Kits specificallyrecognizes the Ser-133 phosphorylated CREB for detection of activated CREB only. Utilization 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 lessthan 5.5 hours. As this assay is performed in 96-well plates, a large number of samples can be
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handled simultaneously, enabling high-throughput automation. This assay is specific for either
total CREB or activated CREB and has been shown to be 4-fold more sensitive and 20-fold fasterthan the gel-retardation technique. With the 5.5-hour TransAM procedure, we could detect CREB
activation using as little as 0.5 µg of nuclear extract from forskolin-stimulated cells. A comparable
assay using EMSA required 2 µg of nuclear extract and a 3-day autoradiography.
TransAM has many applications including the study of drug potency toward CREB-related kinases, CREB transcriptional activity regulation and protein structure/function studies of CREB andits cofactors in areas such as learning, memorization, mental retardation, drug adaptation in thebrain, T-cell repertoire development, hormonal control of metabolic pathways, and many more.

Kit Performance and Benefits
The TransAM CREB Kits are for research use only. Not for use in diagnostic procedures.
Detection limit: <0.5 µg nuclear extract/well.
Range of detection: TransAM provides quantitative results from 0.3 to 10 µg of nuclear extract/well.
Cross-reactivity: TransAM pCREB Kits detect bound phosphorylated CREB-1 (p43) in human,
mouse, rat and hamster extracts. It may also recognize phosphorylated CREM-1 and ATF-1.
TransAM CREB Kits detect total CREB (independent of its phosphorylation state) in human,
mouse, rat and monkey extracts.
pCREB

CREB

Human

+

+

Mouse

+

+

Rat

+

+

Assay time: 3.5 hours.

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TransAM CREB

CREB activation (OD450 nm)

2.5

Untreated WI-38

2

WI-38+Forskolin
1.5
1
0.5
0
0.63

1.25

2.5

5

10

Extract (µg/well)Figure 1: Monitoring CREB activity with the TransAM CREB Kit.
Nuclear extracts from WI-38 cells (Catalog No. 40310) and WI-38 cells stimulated with Forskolin (Catalog No.
40300) were assayed from 0.625-10 µg/well for CREB activation using the TransAM CREB Kit. Data shown are
the results from wells assayed in duplicate. These results are provided for demonstration only.

TransAM pCREB

10.9

pCREB activation (OD 450nm)

0.8

WI-38

0.7

WI-38 + Forskolin

0.6
0.5
0.4
0.3
0.2
0.1
0

0.625

1.25

2.5

5

10

Extract (µg/well)Figure 1: Monitoring pCREB activity with the TransAM pCREB Kit.
Nuclear extracts from WI-38 cells (Catalog No. 40310) and WI-38 cells stimulated with Forskolin (Catalog No.
40300) were assayed from 0.625-10 µg/well for pCREB activation using the TransAM pCREB Kit. Data shown
are the results from wells assayed in duplicate. These results are provided for demonstration only.

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

Quantity
1 plate / 5 plates

Storage

CREB antibody, or
pCREB antibody

10 µl / 50 µl
22 µl / 110 µl-20°C for 6 months-20°C for 6 months

Anti-mouse HRP-conjugated IgG, or
Anti-rabbit HRP-conjugated IgG

11 µl / 55 µl
11 µl / 55 µl

4°C for 6 months
4°C for 6 months

Wild-type oligonucleotide AM2

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

Mutated oligonucleotide AM2

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

WI-38 (Forskolin) nuclear extract

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

Dithiothreitol (DTT)

100 µl / 500 µl(1 M)-20°C for 6 months

Protease Inhibitor Cocktail

100 µl / 500 µl-20°C for 6 months

Herring sperm DNA

100 µl / 500 µl(1 µg/µl)-20°C for 6 months

Lysis Buffer AM1

10 ml / 50 ml

4°C for 6 months

Binding Buffer AM1

10 ml / 50 ml

4°C for 6 months

10X Wash Buffer AM1

22 ml / 110 ml

4°C for 6 months

10X Antibody Binding Buffer AM1

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 CREB assay plate

1/5

4°C for 6 months

Plate sealer

1/5

Room temperature

Reagents

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7

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

8

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 ofphosphorylated CREB 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 of Protease Inhibitor Cocktail per ml of
Lysis Buffer AM1 (see the Quick Chart for Preparing Buffers in this section). Some of the proteaseinhibitors lose their activity after a few hours once diluted. Therefore, we recommend adding
protease inhibitors immediately prior to use. Any remaining Complete Lysis Buffer should bediscarded 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 1 µg/µl Herring sperm DNA per ml of Binding Buffer AM1 (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 AM1 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 AM1 may formclumps, therefore it is necessary to completely resuspend any precipitates by incubating at 50ºC
for 2 minutes and mixing prior to use.
Preparation of 1X Antibody Binding Buffer
Prepare the amount of 1X Antibody Binding Buffer required for the assay as follows: For every
10 ml of 1X Antibody Binding Buffer required, dilute 1 ml 10X Antibody Binding Buffer AM1 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 AM1 may form clumps, therefore it is necessary to completely resuspend
any precipitates by warming to room temperature and vortexing for 1 minute prior to use.

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

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9Preparation of antibodies
Dilute the CREB antibody 1:1000 with 1X Antibody Binding Buffer. Use 100 µl per well. Dilute theanti-mouse HRP-conjugated secondary antibody 1:1000 with 1X Antibody Binding Buffer. Use
100 µl per well.
Dilute the pCREB antibody to 1:500 with the 1X Antibody Binding Buffer. Use 100 µl per well.
Dilute the anti-rabbit HRP-conjugated secondary antibody 1:1000 with 1X Antibody Binding Buffer.
Use 100 µl per well.
Developing Solution
100 µl are needed per well. 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 light during storage. The Developing Solution may develop a yellow hue overtime. This does not affect 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
100 µl are needed per well. Prior to use, transfer the amount of Stop Solution required for theassay into a secondary container (see the Quick Chart for Preparing Buffers in this section). Afteruse, discard remaining Stop Solution.
WARNING:

The Stop Solution is corrosive. Wear personal protective equipment whenhandling, i.e. safety glasses, gloves and labcoat.

WI-38 nuclear extract
The WI-38 (Forskolin) nuclear extract is provided as a positive control for CREB activation. Sufficient extract is supplied for 20 reactions when used at 5 µg/well. We recommend aliquoting
the extract in 5 µl fractions and storing at -80ºC to help avoid multiple freeze/thaw cycles of theextracts. Various nuclear extracts are available from Active Motif.
Wild-type and mutated consensus oligonucleotides
The wild-type consensus oligonucleotide is provided as a competitor for CREB binding in orderto monitor the specificity of the assay. Used at 20 pmol/well, the wild-type oligonucleotide will
prevent CREB binding to the probe immobilized on the plate. Conversely, the mutated consensus
oligonucleotide should have no effect on CREB binding. Prepare the required amount of wildtype and/or mutated consensus oligonucleotide by adding 2 µl of appropriate oligonucleotide to
31.8 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 first, then
add the cell extract. It is not necessary to perform an incubation step of the oligonucleotide in
the well prior to addition of the cell extract. The oligonucleotide competition only needs to be
performed as a control. It is suggested to test the oligonucleotide competition each new cell typeused.

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10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
Herring sperm DNA
Binding Buffer AM1
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 AM1
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 AM1
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

Primary Antibody

CREB antibody
1X Antibody Binding Buffer
TOTAL REQUIRED

0.1 µl112.4 µl112.5 µl

0.9 µl900 µl900 µl5.4 µl5.4 ml5.4 ml10.8 µl
10.8 ml
10.8 mlpCREB antibody
1X Antibody Binding Buffer
TOTAL REQUIRED

0.2 µl112.3 µl112.5 µl

1.8 µl900 µl900 µl10.8 µl
5.4 ml5.4 ml21.6 µl
10.8 ml
10.8 ml

HRP-conjugated anti-mouse IgG
1X Antibody Binding Buffer
TOTAL REQUIRED

0.1 µl112.4 µl112.5 µl

0.9 µl900 µl900 µl5.4 µl5.4 ml5.4 ml10.8 µl
10.8 ml
10.8 ml

HRP-conjugated anti-rabbit IgG
1X Antibody Binding Buffer
TOTAL REQUIRED

0.1 µl112.4 µl112.5 µl

0.9 µl900 µl900 µl5.4 µl5.4 ml5.4 ml10.8 µl
10.8 ml
10.8 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 mlOR

Secondary Antibody
OR* Volumes listed refer to the preparation of buffer for diluting both the primary & secondary antibodies.

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11

CREB 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 Binding Buffer, Wash Buffer,
Antibody Binding Buffer, Developing Solution and Stop Solution into the wells being used.

Step 1: Binding of CREB 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 0.1-10 µg of nuclear extract diluted in Complete Lysis Buffer per well. A protocol
for preparing nuclear extracts can be found on page 15.
Positive control wells: For both CREB and pCREB we recommend adding 5 µg of the provided
nuclear extract diluted in 20 µl of Complete Lysis Buffer per well (2 µl of nuclear extract in 18 µlof Complete Lysis Buffer per well).
Blank wells: Add 20 µl Complete Lysis Buffer only per well.
Reagents

Blank
wells

Positive Control
no competition

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

30 µl
–
–
20 µl
–

30 µl
–
–
–
20 µl

30 µl
–
–
–
20 µl

28 µl
2 µl
–
–
20 µl

28 µl
–
2 µl
–
20 µl

3.

Use the provided adhesive cover to seal the plate. Incubate for 3 hours at room temperature with mild agitation (100 rpm on a rocking platform). Keep any unused wells covered
during the remaining steps in order to preserve those wells for future assays. Any unused
strips from the stripwell plate can be placed in the foil bag, sealed with tape and stored at
4°C.

4.

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

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Step 2: Binding of primary antibody
1.

CREB: Dilute the CREB antibody 1:1000 in 1X Antibody Binding Buffer and mix thoroughly.
pCREB: Dilute the pCREB antibody 1:500 in 1X Antibody Binding Buffer and mix thoroughly.

2.

Add 100 µl of diluted primary antibody to each well being used.

3.

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

4.

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

Step 3: Binding of secondary antibody
1.

CREB: Dilute the HRP-conjugated anti-mouse IgG 1:1000 in 1X Antibody Binding Buffer andmix thoroughly.
pCREB: Dilute the HRP-conjugated anti-rabbit IgG antibody 1:1000 in 1X Antibody Binding
Buffer and mix thoroughly.

2.

Add 100 µl diluted HRP-conjugated antibody to all wells being used.

3.

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

4.

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

5.

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 5-15 minutes at room temperature protected from direct light. Please read the Certificate 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 andpositive 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 an optionalreference wavelength of 655 nm. Blank the plate reader according to the manufacturer’s
instructions using the blank wells.

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13

References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.

Shaywitz A.J. and Greenberg M.E. (1999) Annu. Rev. Biochem. 68: 821-61.
Montminy M.R., Sevarino K.A., Wagner J.A., Mandel G. and Goodman R.H. (1986) Proc. Natl.
Acad. Sci. USA 83: 6682-86.
Hoeffler J.P., Meyer T.E., Yun Y., Jameson J.L. and Habener J.F. (1988) Science 242: 1430-33.
Blendy J.A., Kaestner K.H., Schmid W., Gass P. and Schutz G. (1996) EMBO J. 15: 1098-106.
Hai T.W., Liu F., Coukos W.J. and Green M.R. (1989) Genes Dev. 3: 2083-90.
Foulkes N.S., Borelli E. and Sassone-Corsi P. (1991) Cell 64: 739-49.
Yamamoto K.K., Gonzalez G.A., Biggs W.H. and Montminy M.R. (1988) Nature 334: 494-98.
Kobayashi M. and Kawakami K. (1995) Nucleic Acids Res. 23: 2848-55.
Hai. T and Curran T. (1991) Proc. Natl. Acad. Sci. USA 88: 3720-24.
Gonzalez G.A. and Montminy M.R. (1989) Cell 59: 675-80.
Seamon K.B., Padgett W. and Daly J.W. (1981) Proc. Natl. Acad. Sci. USA 78: 3363-67.
Hagiwara M., Brindle P., Harootunian A., Armstrong R., Rivier J., Vale W., Tsien R. and Montminy M.R. (1993) Mol. Cell. Biol. 13: 4852-59.
Chrivia J.C., Kwok R.P., Lamb N., Hagiwara M., Montminy M.R. and Goodman R.H. (1993)
Nature 365: 855-59.
Silva A.J., Kogan J.H., Frankland P.W. and Kida S. (1998) Annu. Rev. Neurosci. 21: 127-48.
Blendy J.A. and Maldonado R (1998) J. Mol. Med. 76: 104-10.
Hanson R.W. and Reshef L. (1997) Annu. Rev. Biochem. 66: 581-611.
Renard P., Ernest I., Houbion A., Art M., Le Calvez H., Raes M. and Remacle J. (2001) Nucleic
Acids Res. 29: (4) e21.

www.activemotif.com

14

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. Do not use Trypsin toremove cells as it may alter transcription factor activation states.

3.

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

4.

Allow the cells to swell on ice for 15 minutes.

5.

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

6.

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

7.

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

8.

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

9.

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

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15

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.

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16Section B: Troubleshooting Guide
Problem/question

Possible cause

Recommendation

No signal or weak signal

Omission of key
reagent

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

Substrate or conjugate
is no longer active

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

Enzyme inhibitor
present

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

Plate reader settings
not optimal

Verify the wavelength and filter settings

Incorrect assay
temperature

Bring Developing Solution and Stop Solution to room temperature before using

Inadequate volume ofDeveloping Solution

Check to make sure that correct volume is delivered by pipette

Measurement time
too long

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

Concentration of antibodies is too highIncrease antibody dilutions

Inadequate washing

Ensure all wells are filled with Wash Buffer and follow washingrecommendations

Incomplete washingof wells

Ensure all wells are filled with Wash Buffer and follow washingrecommendations

Well cross-contamination

Follow washing recommendations

Too much nuclear
extract per well

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

Concentration of antibodies is too high

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

Not enough nuclear
extract per well

Increase amount of nuclear extract. Not to exceed 50 µg/well

Too many freeze/thaw
cycles of extract

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

CREB is poorly activated or inactivated innuclear fractions

Perform a time course for CREB activation in the studied cell line

Nuclear extracts
are not from correct
species

The CREB and pCREB antibodies work in human, mouse andrat species

Salt concentration toohigh in binding reaction

Reduce amount of extract per well or dialyze extract before use

High background in
all wells

Uneven color
development

High background insample wells

No signal or weak signal
in sample wells

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17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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18