TransAM™
C/EBP α/βTranscription Factor Assay Kits
Catalog Nos. 44196 & 44696(version D3)

Copyright 2017 Active Motif, Inc.

Revision History
Revision

Date

Description of Change

D1

June 2019

Corrected recipes in Quick Chart for Preparing
Buffers

D3

May 2023

Dilution of secondary antibody (HRP-conjugate
anti-mouse IgG) has changed from 1:1,000 to
1:500. Twice as much material has been
provided to account for the change.

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
C/EBP Transcription Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Transcription Factor Assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
TransAM C/EBP α/β. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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
C/EBP 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
The family of C/EBP (CCAAT-ehancer binding protein) basic leucine zipper transcription factors
includes C/EBPα, C/EBPβ, C/EBPδ, C/EBPγ and C/EBPε, all of which exhibit similar DNA-binding
specificities and contain a leucine zipper dimerization region. C/EBP’s play an important role inregulating the balance between cell growth and differentiation. The expression of C/EBP is afunction of tissue type as well as temporal period of development. Therefore, accurate monitoring of C/EBP activity in cells, tissues or animals is crucial for biomedical research and drug
development. To date, such research projects are tedious and time consuming, and lack highthroughput 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 C/EBP α/β Kits are designed specifically for the study of C/
EBP regulated genes. They contain a 96-well plate to which an oligonucleotide containing the C/
EBP consensus binding site has been immobilized. The C/EBPα and C/EBPβ contained in nuclear
extracts specifically binds to this oligonucleotide. By using an antibody that is directed against
either the C/EBPα or C/EBPβ, the C/EBP bound to the oligonucleotide is detected. Addition of asecondary antibody conjugated to horseradish peroxidase (HRP) provides sensitive colorimetricreadout that is easily quantified by spectrophotometry. The 96-well plate with detachable wells
is suitable for manual use or high-throughput screening applications. TransAM C/EBPα/β Kits areavailable in two sizes:

product
TransAM C/EBP α/βformatcatalog no.

1 x 96-well plate5 x 96-well plates

44196
44696

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

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1Flow Chart of Process

3

2

1

5

4

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.
2

1

9

8

7

6

5

4

3

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.
1

5

4

3

2

7

6

A
B
C
D
E
F
G
H

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2

8

9

10

11

12

Introduction
C/EBP Transcription Factor
C/EBPs play an important role in regulating the balance between cell growth and differentiation17.
Expression of C/EBP is, therefore, a function of tissue type as well as temporal period of development1,13.
C/EBPβ is an important transcriptional activator that is involved in the regulation of a number ofgenes related to the immune and inflammatory response and lipid storage. In addition, C/EBPβ is
required for macrophage and B-cell development14. The C/EBPβ regulator also plays a role in the
IFN-signaling pathways and is a mediator of IL-6 signaling11, 15. C/EBPβ expression is enhanced bycytokines during acute phase reactions and may mediate the increase of acute phase proteins. In
other systems, expression of C/EBPβ has been reported to be modulated by growth factor and C/
EBPβ participates in growth hormone-regulated gene expression2. Physiological and functional
interactions of C/EBP with NFκB, Sp1 and other proteins have also been observed7, 8, 12, 16.
There is a temporal order of expression for C/EBPα and C/EBPβ in differentiating cells, and C/
EBPβ is often required for expression of C/EBPα2. While C/EBPβ is induced early during adipocyte
differentiation, C/EBPα is required in the late phase of adipocyte differentiation and up-regulates
the battery of fat-specific genes required for the synthesis, uptake and storage of long chain fatty
acids18.
C/EBPα is a master regulator of myeloid progenitors5, cooperates with other transcription factors
and plays a critical role in early granulocyte development9, 10. Furthermore, C/EBPα is an inhibitorof cell proliferation, and is down-regulated in a large proportion of lung cancers6.
Transcription Factor Assays
To date, three methods are widely used to measure C/EBP expression, either directly or indirectly.
1.

C/EBP expression can be determined by Western blot by using antibodies specific for
C/EBP proteins. This method is time consuming (up to 2 days once the nuclear nuclear
extract are prepared), and is not suitable for processing large numbers of samples.

2.

The DNA-binding capacity of C/EBP 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
C/EBP binding. If C/EBP is active in the nuclear extract, it will bind to the probe. Samples arethen resolved by electrophoresis on a native polyacrylamide gel, followed by autoradiography. This method is sensitive, but like the previous procedure, it is time consuming (multiple
days of gel exposure may be required to achieve sufficient sensitivity) and it cannot beapplied to high-throughput screening. Gelshift assays also require special precautions andequipment for handling radioactivity.

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3

3.

Another method used to assay C/EBP activation is based on reporter genes, typicallyluciferase or β-galactosidase, placed under the control of a promoter containing a C/EBP
consensus binding site. The promoter can be artificial, made of a CCAAT box and a TATA
box, or natural, like the promoter sequences from adipocyte-specific genes, such as thestearoyl-CoA desaturase 1 (SCD1) gene promoter3 or the β3-adrenergic receptor (β3AR) genepromoter4. However, the procedure is limited by the following issues: (i) reporter gene assays have to be repeated several times to obtain statistically reliable data; and (ii) reporter
gene assays are sensitive to confounding factors that may influence the expression level ofthe reporter gene; therefore, assays have to be carefully standardized. Reporter gene assays are sensitive and easy to perform with a large number of samples, but require efficient
cell transfection with the reporter plasmid.

TransAM C/EBP α/β
C/EBP-regulated genes are involved in a variety of cellular pathways that are currently being
deciphered by academic and pharmaceutical laboratories for new target discovery. However, thisfield has been hampered by the lack of convenient assays.
To overcome this, Active Motif is introducing a high-throughput assay to quantify C/EBP activation. TransAM Kits combine a fast and user-friendly ELISA format with a sensitive and specificassay for transcription factors. TransAM C/EBP α/β Kits contain a 96-well plate to which anoligonucleotide that contains a C/EBP consensus binding site (5´-GCAAT-3´) has been immobilized. C/EBP α and β contained in the nuclear extract specifically binds to this oligonucleotide.
The primary antibodies used in the TransAM C/EBP α/β Kit recognize an accessible epitope on C/
EBPα or C/EBPβ protein upon DNA binding. An HRP-conjugated secondary antibody provides asensitive colorimetric readout easily quantified by spectrophotometry. Once the nuclear extracts
are prepared, this assay is completed in less than 3.5 hours. As this assay is performed in 96-wellplates, a large number of samples can be handled simultaneously, enabling high-throughput
automation. This assay is specific for C/EBP α and β 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 C/EBP α and β using as little as 0.5 µg of nuclear extract from rat liver or U-937
cells. A comparable assay using EMSA would require 2.5 µg of nuclear extract and a 3-day
autoradiography.
TransAM C/EBP α/β Kits can be used to examine cell differentiation and development as well as
the regulation of the balance between cell growth and differentiation. Applications include thestudy of C/EBP transcriptional activity regulation, the regulation of lipid storage, Th1 immune
response and macrophage function, the role of growth hormone on C/EBP-regulated gene expression, the regulation and significance of changes in the composition of the C/EBP-containing
protein complex and the study of protein structure/function of C/EBP and its mutated variants.

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4Kit Performance and Benefits
Detection limit: > 0.5 µg nuclear extract per well. TransAM C/EBP α/β is 5-fold more sensitive
than EMSA.
Range of detection: TransAM provides quantitative results from 0.5 to 10 µg of nuclear extract
per well.
Cross-reactivity: Detects C/EBPα from human origin and C/EBPβ from human, mouse, and rat
origin.
Assay time: 3.5 hours. TransAM is 20-fold faster than EMSA.

TransAM C/EBP α/βTransAM C/EBP alpha

C/EBP alpha activation (OD 450nm)

0.7
0.6

Jurkat
U-937

0.5
0.4
0.3
0.2
0.1
0
0.625

1.25

2.5
Extract (µg/well)

5

10

5

10

TransAM C/EBP beta
1.4

C/EBP beta activation (OD 450nm)

1.2
Jurkat
U-937

1
0.8
0.6
0.4
0.2
0

0.625

1.25

2.5
Extract (µg/well)

Monitoring C/EBP activation with TransAM C/EBP α/β Kit.
Nuclear extracts from Jurkat cells (Catalog No. 36014) and U-937 cells (Catalog No. 36030) were assayedfrom 0.625-10 µg per well for C/EBP α or C/EBP β detection using the TransAM C/EBP α/β Kit. Data shown are
the results from wells assayed in duplicate. These results are provided for demonstration only.

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5Kit Components and Storage
TransAM C/EBP α/β Kits are for research use only. Not for use in diagnostic procedures. Except
for the nuclear extract that must be kept at -80°C, kit components can be stored at -20°C 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 whenstored properly.
Reagents

Quantity

Storage

C/EBPα and C/EBPβ antibody

22 µl / 110 µl

4°C for 6 months

HRP-conjugated anti-mouse IgG

24 µl / 120 µl

4°C for 6 months

Wild-type oligonucleotide AM10100 µl / 500 µl(20 pmol/µl)-20°C for 6 months

Mutated oligonucleotide AM10100 µl / 500 µl(20 pmol/µl)-20°C for 6 months

Positive control nuclear extract

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

Dithiothreitol (DTT) (1 M)

100 µl / 500 µl-20°C for 6 months

Protease Inhibitor Cocktail

100 µl / 500 µl-20°C for 6 months

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 C/EBP assay plate

1/5

4°C for 6 months

Plate sealer

1/5

Room temperature

Additional materials required• Nuclear extracts• Multi-channel pipettor• Multi-channel pipettor reservoirs• Rocking platform/orbital shaker• 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. Please refer to the Appendix Section A for a protocol to prepare a nuclear
extract. Our Nuclear Extract Kit can also be purchased separately (Cat. Nos. 40010 & 40410).
When preparing extracts, it is suggested to perform the final lysis step using the Lysis Buffer
AM1 provided in the TransAM Kit. All subsequent dilutions should also be performed using Lysis
Buffer AM1 from the TransAM Kit. Prepare the amount of Complete Lysis Buffer required for theassay by adding 1 µl of 1 M DTT and 10 µl Protease Inhibitor Cocktail per ml of Lysis Buffer AM1(see the Quick Chart for Preparing Buffers in this section). Some of the protease inhibitors lose
their activity after a few hours once diluted. Therefore, we recommend adding protease inhibitors
immediately prior to use. Any remaining Complete Lysis Buffer should be discarded if not used in
the same day.
Preparation of Complete Binding Buffer
Prepare the amount of Complete Binding Buffer AM1 required for the assay by adding 2 μl of DTT
and 20 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
the buffer by warming to room temperature and vortexing for 1 minute prior to use.
Dilute primary antibodies 1:500 and HRP-conjugated secondary antibody to 1:500 with the 1X
Antibody Binding buffer and mix thoroughly.
* 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 U-937 nuclear extract is provided as a positive control for C/EBPα and C/EBPβ detection. This extract
is optimized to give a strong signal when used at 10 µg/well. We recommend aliquoting the extract in
5 µl fractions and storing at -80ºC. Avoid multiple freeze/thaw cycles of the extract. Sufficient extract is
supplied for 10 reactions per plate. 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 C/EBP binding in orderto monitor the specificity of the assay. This competition assay will confirm that the protein
subunits binding to the plate are specific for the C/EBP consensus binding sequence. Used at 40
pmol/well, the oligonucleotide will prevent C/EBP binding to the probe immobilized on the plate.
Conversely, the mutated consensus oligonucleotide should have no effect on C/EBP binding.
Prepare the required amount of wild-type and/or mutated consensus oligonucleotide by adding
2 µl ofappropriate 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 theoligonucleotide 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 type used.

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8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 µl
89 µl
90 µl

0.6 µl5.4 µl534 µl540 µl1.2 µl10.8 µl
1.068 ml
1.08 ml

Complete Binding Buffer

DTT
Herring sperm DNA
Binding Buffer AM1
TOTAL REQUIRED

0.09 µl0.89 µl
44 µl
45 µl

0.7 µl7 µl352 µl360 µl

4.3 µl42.7 µl2.112 ml
2.16 ml

8.6 µl85.5 µl4.224 ml
4.32 ml

Complete Binding Buffer
with C/EBP wild-type ormutated oligonucleotide

C/EBP wild-type or mutated oligo
Complete Binding Buffer
TOTAL REQUIRED

2 µl
43 µl
45 µl

16 µl
334 µl360 µl

96 µl
2.064 ml
2.16 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

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

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9C/EPB Transcription Factor Assay
Read the entire protocol before use.
Determine the appropriate number of microwell strips required for testing samples, controls andblanks in duplicate. Store the unused strips in the aluminum pouch at 4°C. If less than 8 wells in a
strip need to be used, cover the unused wells with a portion of the plate sealer while you performthe assay. The unused wells are stable at room temperature for the duration of the assay if kept
dry. Once the assay is finished, unused strips should be returned to the aluminum pouch andstored at 4°C for a separate assay. Use the strip holder while performing 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 C/EPB to its Consensus Sequence1.

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

2.

Sample wells: Add 10 µl of sample diluted in Complete Lysis Buffer per well. We recommend using 2-20 µg of nuclear extract diluted in Complete Lysis Buffer per well. A protocol
for preparing nuclear extracts is provided on page 13.
Positive control wells: Add 10 µg of the provided U-937 nuclear extract (4 µl of nuclear
extract in 6 µl of Complete Lysis Buffer per well).
Blank wells: Add 10 µl Complete Lysis Buffer only per well.
Reagents

Blank
wells

Positive Control
no competition

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

40 µl
–
–
10 µl
–
–

40 µl
–
–
–
10 µl
–

40 µl
–
–
–
10 µl
–

38 µl
2 µl
–
–
10 µl
–

38 µl
–
2 µl
–
10 µl
–

3.

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

4.

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

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10Step 2: Binding of Primary Antibody1.

Add 100 µl of diluted C/EBPα or C/EBPβ antibody (1:500 dilution in 1X Antibody Binding Buffer) to each well being used, including blank wells.

2.

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

3.

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

Step 3: Binding of Secondary Antibody1.

Add 100 µl of diluted HRP-conjugated antibody (1:500 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 Reaction
13. Remove as much of the final wash as possible by blotting the plate on paper towels.
14. Add 100 µl of room temperature Developing Solution to all wells being used.
15. Incubate under low light conditions from 30 seconds to 5 minutes at room temperature
protected from direct light. Please read the Certificate of Analysis supplied with this kit for
optimal development time associated with this lot number. Monitor the blue color development in the sample wells until they turn medium to dark blue. Do not overdevelop.
16. Add 100 µl of Stop Solution to all the wells. In presence of the acid, the blue color turns
yellow.
17.

Read absorbance on a spectrophotometer within 5 minutes at 450nm with an optionalreference wavelength of 655 nm. Blank the plate reader according to the manufacturer’s
instructions using the blank wells.
Reading the reference wavelength is optional. Most microtiter plate readers are equippedto perform dual wavelength analysis and with the appropriate software, will automaticallysubtract the reference wavelength absorbance from the test wavelength absorbance. If
your plate reader does not have this capability, you may read the plate twice, once at 450
nm and once at 655 nm then manually subtract the 655 nm OD from the 450 nm OD values.

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11

References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.

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Niehof M., Kubicka S., Zender L., Manns M.P. and Trautwein C. (2001) J Mol Biol. 309(4):85568.
Pedersen T.A., Kowenz-Leutz E., Leutz A. and Nerlov C. (2001) Genes Dev. 15(23):3208-16.
Porse B.T., Pedersen T.A., Xu X., Lindberg B., Wewer U.M., Friis-Hansen L. and Nerlov C.
(2001) Cell 107(2):247-58.
Roy S.K., Wachira S. J., Weihua X., Hu J. and Kalvakolanu D.V. (2000) J Biol Chem.
275(17):12626-32.
Ruocco M.R., Chen X., Ambrosino C., Dragonetti E., Liu W., Mallardo M., De Falco
G., Palmieri C., Franzoso G., Quinto I., Venuta S. and Scala G. (1996) J Biol Chem.
1996;271(37):22479-86.
Scott L.M., Civin C.I., Rorth P. and Friedman A.D. (1992) Blood 80(7):1725-35.
Screpanti I., Romani L., Musiani P., Modesti A., Fattori E., Lazzaro D., Sellitto C., Scarpa S.,
Bellavia D. and Lattanzio G. (1995) EMBO J. 14(9):1932-41.
Screpanti I., Musiani P., Bellavia D., Cappelletti M., Aiello F.B., Maroder M., Frati L., ModestiA., Gulino A. and Poli V. (1996) J Exp Med. 184(4):1561-6.
Stein B., Cogswell P.C. and Baldwin A.S. Jr. (1993) Mol Cell Biol. 13(7):3964-74.
Umek R.M., Friedman A.D. and McKnight S.L. (1991) Science 251(4991):288-92.
Yeh W.C., Cao Z., Classon M. and McKnight S.L. (1995) Genes Dev. 9(2):168-81

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

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

1.5 M NaCl

21.9 g

27 mM KCl

0.5 g

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

PIB (Phosphatase Inhibitor Buffer)
125 mM NaF

For 10 ml, mix:
52 mg

250 mM β-glycerophosphate

0.55 g

250 mM p-nitrophenyl phosphate (PNPP)

1.15 g

25 mM NaVO3

31 mg

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

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

For 50 ml, mix:

20 mM Hepes, pH 7.5

0.24 g

5 mM NaF

12 mg

0 µM Na2MoO4

5 µl of a 0.1 M solution

0.1 mM EDTA

10 µl of a 0.5 M solution

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

www.activemotif.com

14Section B: Troubleshooting Guide
Problem/question

Possible cause

Recommendation

No signal or weak signal

Omission of key
reagent

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

Substrate or conjugate
is no longer active

Test conjugate and substrate for activity by mixing a smallaliquot of 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 for primary antibody and 1:5,000
for the secondary antibody. The sensitivity of the assay will bedecreased

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

C/EBP is poorly activated or inactivated innuclear fractions

Perform a time course for C/EBP activation in the studied cell
line

Nuclear extracts
are not from correct
species

The C/EBPα antibody works in human samples, and C/EBPβ
antibodies work in human, mouse and rat samples.

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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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