TransAM™ T-bet(version A)

Catalog Nos. 51396 & 51896

Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, California 92008, USA
Toll free:877 222 9543
Telephone:
760 431 1263
Fax:
760 431 1351
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0800 90 99 79
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Telephone:		+32 (0)2 653 0001
Fax:		
+32 (0)2 653 0050
Active Motif Japan
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Tokyo, 162-0824, Japan
Telephone:+81 3 5225 3638
Fax:
+81 3 5261 8733
Copyright 2008 Active Motif, Inc.

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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
T-bet Transcription Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Transcription Factor Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
TransAM T-bet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Kit Performance and Benefits  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 5Kit Components and Storage
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Protocols
Buffer Preparation and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Quick Chart for Preparing Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
T-bet Transcription Factor Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
References  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Appendix
Section A. Preparation of Nuclear Extract  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Section B. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section C. Related Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  14
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  16

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Overview
Members of the T-box gene family of transcription factors are defined by their homology within
a 200-aa DNA binding domain called the T-box. These proteins are important regulators of several
early developmental processes. Seventeen different T-box genes with diverse regulatory functions
in development and diseases have been identified in mammals. Two T-box genes are expressed incells of the immune system: Eomesodermin and Tbx-21, which is also known as T-bet for T-box
expressed in T-cells. T-bet is a 535 amino acids transcription factor essential to T helper 1 (Th1) cell
generation and effector function. Recently, the expression of T-bet has also been described in
B, natural killer (NK), CD8+ T and dentritic cells. T-bet regulates expression of numerous immune
system-associated genes, including cytokines, cytokine receptors, chemokines, chemokine receptors and cytotoxicity.
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 T-bet Kits are designed specifically to detect and quantify
T-bet activation. They contain a 96-well plate to which oligonucleotide containing the T-bet consensus binding site has been immobilized. T-bet contained in nuclear extract bind specifically tothis oligonucleotide and is detected through use of an antibody directed against T-bet. Additionof 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.

product

formatcatalog no.

TransAM™ T-bet

1 x 96 rxns

51396

TransAM™ T-bet

5 x 96 rxns

51896

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

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1Flow Chart of Process

2

1

7

6

5

4

3

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 .
1

9

8

7

6

5

4

3

2

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 solutionH

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
T-bet Transcription Factor
T-bet (T-box expressed in T-Cells; Tbx21) is a member of the T-box transcription factor family andplays a critical role in the generation of T helper 1 (Th1) cells1. Th1 cells secrete IFN-γ and Tumor
Necrosis Factor β (TNF-β) and are central to cellular immunity against intracellular pathogens. Th1
cells lead to susceptibility to infectious diseases, particularly those of parasites, bacteria, and fungi
for which phagocytosis and intracellular killing are important2. T-bet has been shown to promote
Th1 development, and at least part of its role in this process is due to its ability to directly regulate
key lineage determinant genes such as IFN-γ and CXCR3. T-bet has been shown to bind directly
to the promoter regions of these genes, and the expression of T-bet is required and sufficient to
induce transcription. The level of T-bet expression is increased by signals mediated by the T cell
receptor (TCR). T-bet expression has also been shown to correlate with permissive histone acetylation and methylation, as well as the induction of DNase hypersensitivity at the IFN-γ locus in Th1
cells3.
Transcription Factor Assays
To date, three methods are widely used to measure T-bet activation, either directly or indirectly:
1.

T-bet activation can be determined by Western blot using antibodies specific for T-bet proteins. This method is time consuming (up to 2 days once the nuclear extracts are prepared),and is not suitable for processing large numbers of samples.

2.

The DNA-binding capacity of T-bet can be assayed by gel retardation, also called electrophoretic mobility shift assay (EMSA). In this method, nuclear extracts are incubated with a radioactive double-stranded oligonucleotide probe containing the consensus sequence for T-bet
binding. If T-bet is active in the nuclear extract, it will bind to the probe. Samples are thenresolved by electrophoresis on a native polyacrylamide gel, followed by autoradiography.
This method is sensitive, but like the previous procedure, it is time consuming (multiple daysof gel exposure may be required to achieve sufficient sensitivity) and it cannot be applied tohigh-throughput screening. Gelshift assays also require special precautions and equipment
for handling radioactivity.

3.

Another method used to assay T-bet activation is based on reporter genes, typicallyluciferase or β-galactosidase, placed under the control of a promoter containing the T-box
recognition sequence. The promoter can be artificial and made of several consensus-binding
elements. The procedure is limited by the following issues: (i) reporter gene assays have tobe repeated several times to obtain statistically reliable data; and (ii) reporter gene assays
are sensitive to confounding factors that may influence the expression level of the reporter
gene. Therefore, assays have to be carefully standardized. This method is sensitive and easy
to perform with a large number of samples but requires efficient cell transfection with thereporter plasmid.

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3

TransAM T-bet
The TransAM Kit combines a fast and user-friendly ELISA format with a sensitive and specific assay
for transcription factors. TransAM T-bet Kit contains a 96-well plate on which an oligonucleotide
containing the T-box consensus binding site (5´-AC ACC TAG GTG T-3´) has been immobilized.
The active form of T-bet contained in nuclear extract specifically binds to this oligonucleotide.
The primary antibody used to detect T-bet recognizes an epitope on the T-bet protein upon DNA
binding. Addition of an HRP-conjugated secondary antibody provides a sensitive colorimetricreadout 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-well plates, a large numberof samples can be handled simultaneously, enabling high-throughput automation. This assay is
specific for T-bet activation and has been shown to be 5-fold more sensitive and 20-fold fasterthan the gel-retardation technique. With the 3.5-hour procedure of TransAM, we could detect
T-bet activation using as little as 1 µg of nuclear extract.

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4Kit Performance and Benefits
Detection limit: < 1 µg nuclear extract/well.
Range of detection: TransAM provides quantitative results from 0.125 to 2.5 µg of nuclear extract
per well.
Cross-reactivity: TransAM T-bet specifically detects T-bet from human origin.

T-bet activation (OD 450 nm)

Assay time: 3.5 hours.

2.5

y = 0.8425x + 0.1968

2

R = 0.999

2

1.5
Cos-7 (T-bet transfected)

1

Cos-7

0.5
0

0

1
2
Nuclear Extract (µg)

3Monitoring T-bet activation with the TransAM T-bet Kit:
0.125 to 2.5 µg of Cos-7 (T-bet transfected and treated with PMA/ionomycin) nuclear extract (rectangle-shape) and untransfected Cos-7 nuclear extracts (diamond-shape) were assayed per well. Data shown are the results from wells assayedin duplicate.

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5Kit Components and Storage
TransAM T-bet 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 firstuse. Then, we recommend storing each component at the temperature indicated in the tablebelow. Store the T-bet antibody at 4°C after it has been thawed for use. All components areguaranteed stable for 6 months from date of receipt when stored properly.
Reagents

Quantity
1 plate / 5 plates

StorageT-bet antibody

5 µl / 25 µl

4°C

anti-mouse HRP-conjugated antibody

10 µl / 50 µl

4°C

Wild-type oligonucleotide AM31

100 µl / 500 µl(10 pmol/µl)-20°C

Mutated oligonucleotide AM31

100 µl / 500 µl(10 pmol/µl)

-20°C

Cos-7
nuclear extract (T-bet transfected)
PMA/ionomycin-treated

20 µl / 100 µl(2.5 µg/µl)-80°C

Dithiothreitol (DTT) (1 M)

100 µl / 500 µl-20°C

Protease Inhibitor Cocktail

100 µl / 500 µl-20°C

poly d[I-C]

100 µl / 500 µl(1 µg/µl)-20°C

Lysis Buffer AM1

10 ml / 50 ml

4°C

Binding Buffer AM5

10 ml / 50 ml

4°C

10X Wash Buffer AM2

22 ml / 110 ml

4°C

10X Antibody Binding Buffer AM3

2.2 ml / 11 ml

4°C

Developing Solution

11 ml / 55 ml

4°C

Stop Solution

11 ml / 55 ml

4°C

96-well T-bet assay plate

1/5

Plate sealer

1/5

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. 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). Lysis
Buffer AM1 contains phosphatase inhibitors to prevent dephosphorylation of transcription factors
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 theassay by adding 1 µl of 1 M DTT and 10 µl Protease Inhibitor Cocktail per ml of Lysis Buffer AM1 (seethe Quick Chart for Preparing Buffers in this section). Some of the protease inhibitors lose theiractivity after 24 hours once diluted. Therefore, we recommend using the Complete Lysis Buffer
immediately for cell lysis. The remaining amount should be discarded if not used in the same day.
Preparation of Complete Binding Buffer
Prepare the amount of Complete Binding Buffer AM5 required for the assay by adding 1 μl of DTT
and 10 μl of 17 μg/ml poly[d(l-C)] per ml of Binding Buffer (see the Quick Chart for Preparing Buffers in this section). After use, discard remaining Complete Binding Buffer.
Preparation of 1X Wash Buffer
Prepare the amount of 1X Wash Buffer required for the assay as follows: For every 100 ml of 1X
Wash Buffer required, dilute 10 ml 10X Wash Buffer AM2 with 90 ml distilled water (see the Quick
Chart for Preparing Buffers in this section). Mix gently to avoid foaming. The 1X Wash Buffer may
be stored at 4°C for one week. The Tween 20 contained in the 10X Wash Buffer AM2 may formclumps, therefore homogenize the buffer by incubating at 50ºC for 2 minutes and mixing prior touse.
Preparation of 1X Antibody Binding Buffer
Prepare the amount of 1X Antibody Binding Buffer required for the assay as follows: For every
10 ml of 1X Antibody Binding Buffer required, dilute 1 ml 10X Antibody Binding Buffer AM3 with 9
ml distilled water (see the Quick Chart for Preparing Buffers in this section)*. Mix gently to avoid
foaming. Discard remaining 1X Antibody Binding Buffer after use. The BSA contained in the 10X
Antibody Binding Buffer AM3 may form clumps, therefore homogenize the buffer by warming toroom temperature and vortexing for 1 minute prior to use. Dilute the primary antibody with the
1X Antibody Binding Buffer to 1:2000 for the T-bet and the HRP-conjugated secondary antibody to
1:1000. 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 not affectproduct 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.
Cos-7 nuclear extract (T-bet transfected) PMA/ionomycin-treated
The Cos-7 (T-bet transfected) PMA/ionomycin-treated nuclear extract is provided as a positive
control for T-bet activation. Sufficient extract is supplied for 25 reactions if using 1.25 µg per
well. This extract is optimized to give a strong signal when used at 1.25 µg/well. We recommend
aliquoting the extract in 5 µl fractions and storing at -80ºC. Avoid multiple freeze/thaw cycles ofthe extract.
Wild-type and mutated consensus oligonucleotides
The wild-type consensus oligonucleotide is provided as a competitor for T-bet binding in order tomonitor the specificity of the assay. Used at 40 pmol/well, the oligonucleotide will prevent T-bet
binding to the probe immobilized on the plate. Conversely, the mutated consensus oligonucleotide should have no inhibitory effect on T-bet binding. Prepare the required amount of wild-type
and/or mutated consensus oligonucleotide by adding 4 µl of appropriate oligonucleotide to 36 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
Reagents to prepare
Components
1 well 1 strip 6 strips 12 strips
				(8 wells) (48 wells) (96 wells)
Complete Lysis Buffer

DTT
Protease Inhibitor Cocktail
Lysis Buffer
Total Required

0.01 µl 0.1 µl
0.6 µl0.12 µl 0.9 µl
5.4 µl11.12 µl 89.0 µl 534.0 µl
11.25 µl 90.0 µl 540.0 µl

1.2 µl10.8 µl1.07 ml
1.08 ml

Complete Binding Buffer DTT
poly d[I-C]
Binding Buffer
Total Required

0.04 µl 0.3 µl
0.45 µl 3.6 µl
44.5 µl 356.1 µl
45 µl 360 µl

2.16 µl 4.3 µl
21.6 µl 43.2 µl
2.14 ml 4.27 ml
2.16 ml 4.32 ml

Binding Buffer with
T-bet wt or
mut oligont

wt or mut oligont
Complete Binding Buffer
Total Required

4 µl
32 µl 192 µl
41 µl 328 µl
1.97 ml
45.0 µl 360.0 µl 2.16 ml

1X Washing Buffer

Distilled Water
10X Washing Buffer
Total Required

2.025 ml 16.2 ml 97.2 ml 194.4 ml
225.0 µl
1.8 ml 10.8 ml 21.6 ml
2.25 ml 18.0 ml 108.0 ml 216.0 ml

1X Antibody
Binding Buffer*

Distilled Water
202.5 µl
1.62 ml 9.72 ml 19.44 ml
10X Antibody Binding Buffer 22.5 µl 180.0 µl
1.08 ml 2.16 ml
Total Required
225.0 µl
1.8 ml 10.8 ml 21.6 ml

Developing Solution

Total Required

112.5 µl 900.0 µl

5.4 ml 10.8 ml

Stop Solution

Total Required

112.5 µl 900.0 µl

5.4 ml 10.8 ml

N/A
N/A
N/A

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

T-bet 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 with aportion 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. Multichannel 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.

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9

Step 1: Binding of T-bet 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 (4 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 2-10 µg of nuclear extract diluted in Complete Lysis Buffer per well. A protocol for
preparing nuclear extracts is provided on page 11.
Positive control wells: Add 1.25 µg of the provided nuclear extract diluted in 10 µl of Complete Lysis Buffer per well (0.5 µl of nuclear extract in 9.5 µl of Complete Lysis Buffer perwell).
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 a sinkto empty the wells, then tap the inverted plate 3 times on absorbent paper towels.

Step 2: Binding of primary antibody1.

Add 100 µl diluted T-bet antibody (1:2000 dilution in 1X Antibody Binding Buffer) to each wellbeing 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 of diluted HRP-conjugated antibody (1:1000 dilution in 1X Antibody Binding Buffer)to all wells being used.

2.

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

3.

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

4.

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

Step 4: Colorimetric reaction1.

Add 100 µl Developing Solution to all wells being used.

2.

Incubate 2-15 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.

Beima, K.M., et al. (2006) J Biol. Chem, 281: 11992-12000.
Tong, Y., et al.. (2005) PNAS, 102: 2034-2039.
Lewis, M.D., et al. (2007) Mol Cell Biol, 27:8510-8521.

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 theassay that may arise from using homemade or other buffers. If you prefer to make your own buffers, please refer to the following protocol.
This procedure can be used for a confluent cell layer of 75 cm2 (100-mm dish). The yield is approximately 0.15 mg of nuclear proteins for 9 x 106 cells.
1.

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

2.

Add 10 ml ice-cold PBS/PIB and scrape the cells off the dish with a cell lifter. Transfer cells
into a pre-chilled 15 ml tube and spin at 300 x g for 5 minutes at 4°C.

3.

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

4.

Allow the cells to swell on ice for 15 minutes.

5.

Add 50 µl 10% Nonidet P-40 (0.5 % final) and vortex the tube vigorously for 10 seconds.

6.

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

7.

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

8.

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

9.

Determine the protein concentration of the extract by using a Bradford-based assay.

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11Preparation of Buffers for Nuclear Extract
10X PBS				For 250 ml, mix:
0.1 M phosphate buffer, pH 7.5		
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. Storethe 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

10 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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12Section B: Troubleshooting Guide
Problem/question

Possible cause

Recommendation

No signal or weak signal

Omission of key
reagent

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

Substrate or conjugate
is no longer active

Test conjugate and substrate for activity

Enzyme inhibitor
present

Sodium azide will inhibit the peroxidase reaction, follow ourrecommendations to prepare buffers

Plate reader settings
not optimal

Verify the wavelength and filter settings in the plate reader

Incorrect assay temperature

Bring substrate to room temperature

Inadequate volume ofDeveloping Solution

Check to make sure that correct volume is delivered by pipette

Developing time too
long

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

Concentration of antibodies is too highIncrease antibody dilutions

Inadequate washing

Ensure all wells are filled with Wash Buffer and follow washingrecommendations

Incomplete washingof wells

Ensure all wells are filled with Wash Buffer and follow washingrecommendations

Well cross-contamination

Follow washing recommendations

High background in all
wells

Uneven color development

High background in sample Too much nuclear
wells
extract per well

No signal or weak signal insample wells

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 1:2000 for primary antibody and
1:5000 for the secondary antibody. The sensitivity of the assay
will be decreased

Not enough nuclear
extract per well

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

Perform a time course for T-bet activation in the studied cell
line

Nuclear extracts are
not from correct
specieswww.activemotif.com

13Section C. Related Products
Transcription Factor ELISAs		FormatCatalog No.

TransAM™ AML-1/Runx1		
TransAM™ AML-3/Runx2		
TransAM™ AP-1 Family		TransAM™ AP-1 c-Fos		
TransAM™ AP-1 c-Jun		
TransAM™ AP-1 FosB		
TransAM™ AP-1 JunD		
TransAM™ ATF-2		
TransAM™ c-Myc		
TransAM™ C/EBP a/b		
TransAM™ CREB		
TransAM™ pCREB		
TransAM™ Elk-1		
TransAM™ ER		TransAM™ FKHR (FOXO1/4)		
TransAM™ GATA Family		TransAM™ GATA-4		
TransAM™ GR		TransAM™ HIF-1		
TransAM™ HNF Family		TransAM™ HNF-1		
TransAM™ IRF-3 (Human)		
TransAM™ IRF-3 (Mouse)		
TransAM™ IRF-7 		
TransAM™ MAPK Family		TransAM™ MEF2		
TransAM™ MyoD		
TransAM™ NF-YA		
TransAM™ NFATc1		
TransAM™ NFkB Family		TransAM™ Flexi NFkB Family		TransAM™ NFkB p50		
TransAM™ Flexi NFkB p50		
TransAM™ NFkB p50 Chemi		
TransAM™ NFkB p52		
TransAM™ NFkB p52 Chemi		
TransAM™ NFkB p65		
TransAM™ Flexi NFkB p65		
TransAM™ NFkB p65 Chemi		
TransAM™ Oct-4		
TransAM™ p53		
TransAM™ PPARg		
TransAM™ Sp1		
TransAM™ Sp1/Sp3		
TransAM™ STAT Family		TransAM™ STAT3		

1 x 96-well plate1 x 96-well plate2 x 96-well plates
1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate2 x 96-well plates
1 x 96-well plate1 x 96-well plate1 x 96-well plate2 x 96-well plates
1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate2 x 96-well plates
1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate2 x 96-well plates
2 x 96-well plates
1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate2 x 96-well plates
1 x 96-well plate

47396
44496
44296
44096
46096
45096
43496
42396
43396
44196
42096
43096
44396
41396
46396
48296
46496
45496
47096
46296
46196
48396
48496
50196
47296
43196
47196
40396
40296
43296
43298
41096
41098
41097
48196
48197
40096
40098
40097
41196
42496
40196
41296
40496
42296
45196

www.activemotif.com

14

Chromatin Assembly		

FormatCatalog No.

Chromatin Assembly Kit		

10 rxns

53500

Histone Acetyltransferase Activity		FormatCatalog No.

HAT Assay Kit (Fluorescent)		

1 x 96 rxns

56100

Histone Purification		

FormatCatalog No.

Histone Purification Kit		

10 rxns

40025

Co-Immunoprecipitation		

FormatCatalog No.

Nuclear Complex Co-IP Kit		
Universal Magnetic Co-IP Kit		

50 rxns
25 rxns

54001
54002

DNA Methylation		

FormatCatalog No.

MethylDetector™		
MethylCollector™		

50 rxns
25 rxns

55001
55002

SUMOylation		

FormatCatalog No.

SUMOlink™ SUMO-1 Kit		
SUMOlink™ SUMO-2/3 Kit		

20 rxns
20 rxns

40120
40220

www.activemotif.com

15

Technical Services
If you need assistance at any time, please call Active Motif Technical Service at one of thenumbers listed below.
Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008
USA
Toll Free:877 222 9543
Telephone: 760 431 1263
Fax:
760 431 1351
E-mail:tech_service@activemotif.com
Active Motif Europe
104 Avenue Franklin Roosevelt
B-1330 Rixensart, Belgium
UK Free Phone:		
0800 169 31 47
France Free Phone:
0800 90 99 79
Germany Free Phone: 0800 181 99 10
Telephone: 		+32 (0)2 653 0001
Fax: 		
+32 (0)2 653 0050
E-mail: 		
eurotech@activemotif.com
Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Telephone: +81 3 5225 3638
Fax:
+81 3 5261 8733
E-mail:japantech@activemotif.com
Visit Active Motif on the worldwide web at http://www.activemotif.com
At this site:• Read about who we are, where we are, and what we do• Review data supporting our products and the latest updates• Enter your name into our mailing list to receive our catalog, MotifVations newsletter andnotification of our upcoming products• Share your ideas and results with us• View our job opportunities
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www.activemotif.com

16