TransAM™ FKHR

Transcription Factor Assay Kits
(version C2)
Catalog Nos. 46396 & 46896

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.
© 2008 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
FKHR Transcription Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Transcription Factor Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
TransAM FKHR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Kit Performance and Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Protocols
Buffer Preparation and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Quick Chart for Preparing Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
FKHR Transcription Factor Assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Appendix
Section A: Preparation of Nuclear Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Section B: Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

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Overview
The Forkhead transcription factor FKHR regulates expression of gluconeogenic factors, andplays a key role in insulin regulation of glucose levels. Besides its role in glucose homeostasis,
FKHR has been shown to be involved in pathological processes such as type 2 diabetes, cancer
and organismal aging. The transcriptional output of FKHR is controlled by a two-tiered mechanism of phosphorylation and acetylation, and alterations in this balance can result in profoundeffects.1 Therefore, accurate monitoring of FKHR activity in cells, tissues or animals is crucial forbiomedical research and drug development. 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 FKHR Kits are designed specifically for the study of
FKHR. They contain a 96-well plate to which a Forkhead Box (FOX) element has been immobilized. FKHR contained in nuclear extracts binds specifically to this DNA molecule and is detected
through use of an antibody directed against FKHR. Addition of a secondary antibody conjugatedto 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 FKHR Kits are available in two sizes:

product
TransAM FKHR

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formatcatalog no.

1 x 96-well plate5 x 96-well plates

46396
46896

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

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9

10

11

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Introduction
FKHR Transcription Factor
The Forkhead family of transcription factors are involved in regulation of the cell cycle, cell death,cell metabolism and oxidative stress (see 2 for review). The FOXO (Forkhead Box, class O) proteins form a subfamily of Forkhead transcription factors that are direct targets of phosphoinositide 3-kinase (PI3K) mediated signal transduction.3 In the presence of growth factor/survival
signals, PI3K activation leads to PDK mediated downstream activation of PKB/c-AKT. Phosphorylation and activation of PKB causes nuclear translocation of PKB, which phosphorylates and inactivates nuclear FOXO. FOXO then binds 14-3-3 proteins, and the FOXO/14-3-3 complex is exported
to the cytoplasm. Phosphorylated, inactive FOXO proteins remain bound to 14-3-3 proteins in
the cytoplasm, thereby preventing nuclear import of FOXO. In the absence of survival signals,
cytoplasmic FOXO is dephosphorylated, causing dissociation from 14-3-3 proteins and allowing
nuclear import of FOXO to activate gene expression.3
There are three AKT phosphorylation sites in the FKHR protein: Thr24, Ser256 and Ser319.4 Specifically, phosphorylation at Ser256 is thought to play a role in masking a FKHR nuclear localization signal.5 In addition, phosphorylation at Ser256 may also mediate the effects of insulin on
gene expression.6 Phosphorylation at Thr24 is critical for FKHR interaction with 14-3-3 proteins.5
The most well studied FOXO members include acute-lymphocytic-leukaemia-1 fused gene fromchromosome X (AFX/FOXO4), Forkhead in rhabdomyosarcoma (FKHR/FOXO1) and FKHR-Like 1(FKHRL1/FOXO3a). AFX mRNA is expressed at high levels in heart and skeletal muscle, and moderately in brown and white adipose tissue. FKHR mRNA is detected at its highest levels in brown
adipose tissue, white adipose tissue and spleen, with lower levels in liver and skeletal muscle.
FKHRL1 mRNA is expressed at its highest levels in brain, heart, kidney and spleen, with moderate
expression in white adipose tissue and testis.7
In different cell types, FOXO proteins modulate various cellular activities. In hepatocytes, FOXO
proteins regulate the expression of factors involved in gluconeogenesis, such as peroxisome
proliferators-activated receptor-γ coactivator-1, glucose-6-phosphate and phosphoenolpyruvatecarboxykinase .8-11 Using genetic gain and loss of function analysis in mice, FKHR has also beenshown to control β cell compensation for insulin resistance and glucose production in type 2
diabetes.12 In addition, FOXO proteins such as FKHRL1 have been shown to regulate catalase andsuperoxide dismutase gene expression that protect cells from oxidative stress, suggesting that
FOXO factors act to control the mammalian lifespan.13, 14
While Forkhead transcription factors do not bind to a clear consensus sequence, the subclasses
have been shown to bind to specific sequence elements. For example, FOXO and HNF subclassesof Forkhead proteins have been shown to bind to the insulin response elements (IREs) of insulinlike growth factor-binding protein-1 (IGFBP-1).11 FOXO transcription factors bind to a consensus
core sequence of 5´-TTGTTTAC-3´15, which includes a sequence TRTTTAY (with R a purine baseand Y a pyrimidine base) conserved among various Forkhead members.7
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3

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

Expression can be measured by Western blot, using antibodies raised against FKHR. Thismethod is time consuming (up to 2 days once the nuclear extracts are prepared), and is notsuitable for processing large numbers of samples.

2.

The DNA-binding capacity of FKHR 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 a specific sequence elementfor FKHR binding. If FKHR is active in the nuclear extract, it will bind to the probe. Samples
are then 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 be applied to high-throughput screening. Gelshift assays also require special precautions and equipment for handling radioactivity.

3.

Another method used to assay FKHR activation is based on reporter genes, typically luciferase or β-galactosidase, placed under the control of a promoter containing a FKHR consensus binding element. This promoter can be artificial, made of several FKHR cis-elements
and a TATA box, or natural. Limitations of this procedure are: (i) reporter gene assays haveto be repeated several times to obtain statistically reliable data; and (ii) reporter geneassays are sensitive to confounding factors that may influence the expression level of thereporter gene. Therefore, assays have to be carefully standardized. This method is sensitiveand easy to perform with a large number of samples but requires efficient cell transfection
with the reporter plasmid.

TransAM FKHR (FOXO1)FKHR is involved in cell survival and regulation of glucose levels, and therefore represents an
excellent pharmacological target for developing drugs to treat cancer and diabetes. However,pharmaceutical research in this field has been limited by the lack of convenient assays suitablefor processing large numbers of samples.
To overcome this, Active Motif is introducing a high-throughput assay to quantify FKHR activation. The TransAM Kit combines a fast and user-friendly ELISA format with a sensitive andspecific assay for transcription factors. TransAM FKHR (FOXO1) Kits contain a 96-well plate on
which has been immobilized oligonucleotide that contains a FOXO element. The active formof FKHR contained in the nuclear extract specifically binds to this oligonucleotide. The primary
antibodies used to detect FKHR will recognize an epitope on FKHR protein that is accessible only
when FKHR is activated and bound to the target DNA. Addition of a secondary HRP-conjugatedantibody provides a sensitive colorimetric readout easily quantified by spectrophotometry. Oncethe nuclear extracts are prepared, this assay is completed in less than 3.5 hours. As this assay is
performed in 96-well plates, a large number of samples can be handled simultaneously, enablinghigh-throughput automation. With the TransAM procedure, activated FKHR was detected in 0.5µg of nuclear extract.
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4Kit Performance and Benefits
The TransAM FKHR Kit is for research use only. Not for use in diagnostic procedures.
Detection limit: <5 µg nuclear extract/well.
Range of detection: TransAM provides quantitative results from 5 to 20 µg of nuclear extract/well.
Cross-reactivity: TransAM FKHR detects FKHR (FoxO1) from human, mouse and rat origin.
Assay time: 3.5 hours.

FKHR activation (OD 450 nm)

2.0
1.5
1.0
0.5
0.0

1.2

2.5

5

10

20

Nuclear extract (µg)-0.5Figure 1: Monitoring FKHR activity with the TransAM FKHR Kit.
Different amounts of nuclear extracts from untreated Raji (dark bars) and HeLa (light bars) cells are tested for
FKHR activity by using the TransAM FKHR Kit. This data is provided for demonstration only.

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5Kit Components and Storage
Except for the nuclear extract that must be kept at -80ºC, kit components can be stored at -20ºCprior to first use. Then, we recommend storing each component at the temperature indicated in
the table below.
Quantity
1 plate / 5 plates

Storage / Stability

FKHR (FOXO1) antibody

12 µl / 60 µl-20°C for 6 months

Anti-rabbit HRP-conjugated IgG

11 µl / 55 µl(0.2 µg/µl)

4°C for 6 months

Wild-type oligonucleotide AM19100 µl / 500 µl(10 pmol/µl)-20°C for 6 months

Mutated oligonucleotide AM19100 µl / 500 µl(10 pmol/µl)-20°C for 6 months

Raji 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 AM6

10 ml / 50 ml

4°C for 6 months

10X Wash Buffer AM2

22 ml / 110 ml

4°C for 6 months

10X Antibody Binding Buffer AM2

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 FKHR assay plate

1/5

4°C for 6 months

Plate sealer

1/5

Reagents

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

6

Protocols
Buffer Preparation and Recommendations
Preparation of Complete Lysis Buffer
We provide an excess of Lysis Buffer AM1 in order to perform the assay AND to prepare customized nuclear extracts. Our Nuclear Extract Kit can also be purchased separately (Cat. Nos. 40010& 40410). 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 protease inhibitors lose their activity after 24 hours
once diluted. Therefore, we recommend using the Complete Lysis Buffer immediately for cell
lysis. The remaining amount should be discarded if not used in the same day.

Preparation of Complete Binding Buffer
Prepare the amount of Complete Binding Buffer required for the assay by adding 10 µl of 1 µg/µl Herring sperm DNA per ml of Binding Buffer AM6 (see the Quick Chart for Preparing Buffers inthis section). After use, discard remaining Complete Binding Buffer.

Preparation of 1X Wash Buffer
Prepare the amount of 1X Wash Buffer required for the assay as follows: For every 100 ml of 1X
Wash Buffer required, dilute 10 ml 10X Wash Buffer AM2 with 90 ml distilled water (see the Quick
Chart for Preparing Buffers in this section). Mix gently to avoid foaming. The 1X Wash Buffer may
be stored at 4°C for one week. The Tween 20 contained in the 10X Wash Buffer AM2 may formclumps, therefore homogenize the buffer by 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 AM2 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 AM2 may form clumps, therefore homogenize the buffer by warming toroom temperature and vortexing for 1 minute prior to use. Dilute the primary antibody to 1:1000
and the secondary antibody to 1:1000 with the 1X Antibody Binding Buffer. Depending on the particular assay, the signal:noise ratio may be optimized by using higher dilutions of both antibodies.
This may decrease the sensitivity of the assay.
* Volumes listed refer to the preparation of buffer for diluting both the primary & secondary antibodies.

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7Developing Solution
The Developing Solution should be warmed to room temperature before use. The Developing
Solution is light sensitive, therefore, we recommend avoiding direct exposure to intense lightduring storage. The Developing Solution may develop a yellow hue over time. This does notaffect product performance. A blue color present in the Developing Solution indicates that it has
been contaminated and must be discarded. Prior to use, place the Developing Solution at room
temperature for at least 1 hour. Transfer the amount of Developing Solution required for the assay
into a secondary container before aliquoting into the wells (see the Quick Chart for Preparing
Buffers in this section). After use, discard remaining Developing Solution.

Stop Solution
Prior to use, transfer the amount of Stop Solution required for the assay into a secondary
container (see the Quick Chart for Preparing Buffers in this section). After use, discard remaining
Stop Solution.
WARNING:

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

Raji nuclear extract
The Raji nuclear extract is provided as a positive control for FKHR activation. Sufficient extract
is supplied for 20 reactions. This extract is optimized to give a strong signal 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 the extracts. Various nuclear extracts are available from Active
Motif (see Appendix, Section B. Related Products).

Wild-type and mutated consensus oligonucleotides
The wild-type consensus oligonucleotide is provided as a competitor for FKHR binding in order tomonitor the specificity of the assay. Used at 10 pmol/well, the oligonucleotide will prevent FKHR
binding to the probe immobilized on the plate. Conversely, the mutated consensus oligonucleotide should have no effect on FKHR binding. Prepare the required amount of wild-type and/or
mutated consensus oligonucleotide by adding 1.13 µl of appropriate oligonucleotide to 43.88 µlof Complete Binding Buffer per well being used (see the Quick Chart for Preparing Buffers in thissection). To allow for optimum competition, add the oligonucleotide to the well prior to additionof the nuclear extract.

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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 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

Herring Sperm DNA
Binding Buffer AM6
TOTAL REQUIRED

0.45 µl44.55 µl
45 µl

3.6 µl356.4 µl
360 µl21.6 µl2.138 ml
2.16 ml43.2 µl
4.277 ml
4.32 ml

Complete Binding Buffer
with wild-type ormutated oligonucleotide

Wild-type or mutated oligo
Complete Binding Buffer
TOTAL REQUIRED

1.13 µl43.88 µl
45 µl

9 µl
351 µl360 µl54 µl2.106 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 AM2
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.

FKHR 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.

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9

Step 1: Binding of FKHR 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 10 pmol (1 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 11.
Positive control wells: Add 5 µg of the provided nuclear extract diluted in 10 µl of Complete
Lysis Buffer per well (2 µl of nuclear extract in 8 µl of Complete Lysis Buffer per well).
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 Wash Buffer. For each wash, flick the plate over asink to empty the wells, then tap the inverted plate 3 times on absorbent paper towels.

Step 2: Binding of primary antibody1.

Add 100 µl diluted FKHR antibody (1:1000 dilution in 1X Antibody Binding Buffer) to eachwell being used.

2.

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

3.

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

Step 3: Binding of secondary antibody1.

Add 100 µl diluted HRP-conjugated antibody (1:1000 dilution in 1X Antibody Binding Buffer)to all wells being used.

2.

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

3.

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

4.

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

Step 4: Colorimetric reaction1.

Add 100 µl 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.

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

Accili D. and Arden K.C. (2004) Cell 117: 421-426.
Burgering B.M.T. and Kops G.J.P.L. (2002) TIBS 27(7): 352-360.
Birkenkamp K.U. and Coffer P.J. (2003) Bioch. Soc. Trans. 31(1): 292-296.
Woods Y.L. et al (2001) Biochem J. 355: 597-607.
Rena G. et al (2001) Biochem J. 354: 605-612.
Guo S. et al (1999) J. Biol. Chem. 274: 17184-17192.
Pierrou S. et al (1994) EMBO J. 13(20): 5002-12.
Daitoku H. et al (2003) Diabetes 52(3): 642-9.
Schmoll D. et al (2000) J. Biol. Chem. 275(46): 36324-33.
Ayala J.E. et al (1999) Diabetes 48(9): 1885-9.
Hall R.K. et al (2000) J. Biol. Chem. 29;275(39): 30169-75.
Nakae J. et al (2002) Nature Genetics 32(2): 245-53.
Nemoto S. et al (2002) Science 295(5564): 2450-2.
Kops G.J. et al (2002) Nature 419(6904): 316-21.
Furuyama T. et al (2000) Biochem J. 349(Pt 2): 629-34.

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11

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 0.5 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 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

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12

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.
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.

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13Section B: 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

High background in all wells

Uneven color development

High background in sample wells

No signal or weak signal insample wells

www.activemotif.com

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
2.5 µg/well

Concentration of antibodies
too high

Perform antibody titration to determine
optimal working concentration. Start using
1:1000 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

FKHR is poorly activated orinactivated

Perform a time course for FKHR activationin the studied cell line

Samples are not fromcorrect species

Refer to cross-reactivity information on page 5

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