TransAM™ STAT Family

Transcription Factor Assay Kit(version E3)
Catalog No. 42296

Copyright 2013 Active Motif, Inc.

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.
© 2013 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
STAT Transcription Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Transcription Factor Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
TransAM STAT Family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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
STAT Family Transcription Factor Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Appendix
Section A. Preparation of Nuclear Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Section B. Troubleshooting Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14

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Overview
Signal transducers and activators of transcription (STAT) proteins are latent transcription factorsthat are activated by phosphorylation via tyrosine kinases. Over 35 different extracellular polypeptides activate Janus kinase associated receptors, leading to phosphorylation of Janus kinases
and the subsequent phosphorylation of STAT proteins. Upon phosphorylation, the STAT proteins
dimerize and migrate to the nucleus where they exert transcriptional activation. Phosphorylation of a single tyrosine localized around residue 700 is crucial for activation of each STAT family
member1. STAT proteins are involved in a wide variety of biological pathways. STAT1 is involvedin the activation of IFNα and IFNγ genes, STAT2 in the activation of IFNα genes, STAT4 in T-helper
cell development and STAT5 in milk production. Disruption of STAT functions in mouse leads toseveral defects such as immune deficiency (STAT1), embryonic lethality (STAT2), lack of gastrulation (STAT3), T-helper 1 cell dysfunction (STAT4), lack of lactation (STAT5A, 5B) and T-helper 2cell dysfunction (STAT6). The disruption of STAT signaling blocks neoplastic transformation, thus
making inhibitors of STAT proteins candidates for the treatment of cancer. Therefore, accuratemonitoring of STAT activity in cells, tissues or animals is crucial for biomedical research and drug
development.
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 STAT Family Kits are designed specifically for the studyof STAT pathways. They contain a 96-well plate to which oligonucleotide containing a STAT consensus binding site has been immobilized. STATs contained in nuclear extracts binds specificallyto this oligonucleotide and are detected through use of an antibody directed against STAT 1α, 3,5A or 5B. Addition of a secondary antibody conjugated to horseradish peroxidase (HRP) provides
sensitive colorimetric readout that is easily quantified by spectrophotometry. The 96-well platewith individual strips of 8 wells is suitable for manual use or high-throughput screening applications:product
TransAM™ STAT Family

formatcatalog no.

2 x 96 rxns

42296

* Technology covered by AAT-filed patents and licensed to Active Motif.
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1Flow Chart of Process

1

5

4

3

2

7

6

8

9

10

11

12

A
B
C
D
E

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.

9

8

7

6

5

4

3

2

1

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.
5

4

3

2

1

7

6

A
B
C
D
E
F
G
H

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2

8

9

10

11

12

Introduction
STAT Transcription Factor
STAT (signal transducers and activators of transcription) transcription factors were discovered
fourteen years ago as mediators of interferon-induced gene expression. They comprise a familyof latent cytoplasmic proteins that are activated to participate in gene control when cells encounter various extracellular polypeptides. Their critical role in development and normal cell signaling
has been largely determined through the analysis of transgenic mice lacking individual STAT
genes. The STAT family consists of seven members that are activated by virtually every cytokine
and growth factor (Table 1).
FactorMass (kDa)

STAT1

91

IFNα, IFNγ, EGF, PDGF, FGF, ACRII,

1

STAT2

113

IFNα/β (with STAT1)

1

STAT3

92

IL-6, LIF, CNTF, OM, CT-1, EGF, G-CSF, IL-10, leptin

1, 2, 3

STAT4

89

IL-12

2, 4

STAT 5A, 5B 77, 80

IL-2, IL-3, IL-5, IL-7, IL-15, GM-CSF, Prolactin, Epo

5, 6

STAT6

IL-4, IL-13

7, 8

94

Activating Cytokine

Reference No.

The STAT proteins are unique among transcription factors in containing an SH2 (src-homology 2),
phosphotyrosine-binding domain, a common protein-protein interaction domain among signaling proteins9. Tyrosine phosphorylation around residue 700 is essential for the dimerization of
STATs and the concomitant nuclear translocation of the dimer. Ligand-activated receptors thatcatalyze this phosphorylation include receptors with intrinsic tyrosine kinase activity (epidermal
growth factor (EGF), platelet-derived growth factor (PDGF) and colony-stimulating factor-1) as
well as receptors that lack intrinsic tyrosine kinase activity but to which Janus kinases (JAKs)
are noncovalently associated10, 11. Receptors to which JAKs are bound are often referred to ascytokine receptors. Their ligands include IFN-α, -β and -γ; interleukins (IL) 2 to 7, 10 to 13, and 15;
and erythropoietin, growth hormone, prolactin, thrombopoietin and other polypeptides. STAT
dimers and heterodimers, but not monomers, are competent to bind DNA. The known DNA binding heterodimers are STAT1:2 (strong binding requires the joint presence of another protein, p48)
and STAT1:312. STATs that form homodimers that bind DNA include STAT 1, 3, 4, 5 (STAT5A and 5B
interact in a manner equivalent to a heterodimer) and 6 10, 11, 12.
In most cases, STAT activation is transient. Inactivation of STAT proteins is carried out by several
mechanisms, including dephosphorylation of STAT proteins in the nucleus and degradation
through the ubiquitin-proteosome pathway13. A novel family of negative feedback inhibitors of the
JAK-STAT pathway has been identified, referred to as suppressor-of-cytokine-signaling (SOCS)
proteins/JAK binding (JAB) proteins, and STAT-induced STAT inhibitors (SSIs)14, 15, 16. In addition, afamily of protein inhibitors of activated STAT (PIAS) proteins has been identified17, 18.

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3

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

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

2.

The DNA-binding capacity of STAT 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
STAT binding. If STAT 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.

3.

Another method used to assay STAT activation is based on reporter genes, typicallyluciferase or β-galactosidase, placed under the control of a promoter containing the STAT
consensus sequence. This promoter can be artificial, made of several STAT cis-elements
and a TATA box, or natural, like the HIV long terminal repeat (LTR) sequence. Limitations of
this procedure are: (i) reporter gene assays have to be repeated several times to obtainstatistically reliable data; and (ii) reporter gene assays are sensitive to confounding factorsthat may influence the expression level of the reporter gene. Therefore, assays have to becarefully standardized. This method is sensitive and easy to perform with a large number ofsamples but requires efficient cell transfection with the reporter plasmid.

TransAM STAT Family
STAT transcription factors convert extracellular stimuli into diverse biological responses such as
the regulation of development, cell proliferation, differentiation and apoptosis. Aberrant STAT
signaling can affect the outcome of these pathways and result in oncogenesis. Discovery anddevelopment of novel inhibitors of STAT signaling hold significant promise for providing more
effective treatment for a wide variety of cancers at various stages of malignant progression.
To help achieve this, Active Motif offers a high-throughput assay to quantify STAT 1α, 3, 5A and 5B
activation7. The TransAM Kit combines a fast and user-friendly ELISA format with a sensitive andspecific assay for transcription factors. TransAM STAT Family Kits contain a 96-well plate on which
has been immobilized oligonucleotide containing the STAT consensus binding site (5´-TTCCCGGAA-3´). The active form of STAT contained in nuclear extract specifically binds to this oligonucleotide. The primary antibodies used to detect STAT recognize an epitope on STAT 1α, 3, 5A or 5B
that is accessible only when STAT is activated and bound to its target DNA. The anti-STAT1α antibody recognizes only the alpha subunit of human STAT1, the anti-STAT3 antibody recognizes boththe alpha and beta forms of human STAT3, and the anti-STAT5A and 5B antibodies recognize boththe alpha and beta forms of human STAT5A and STAT5B. An HRP-conjugated secondary antibody
provides a sensitive colorimetric readout that is easily quantified by spectrophotometry. Once
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4the nuclear extract is prepared, this assay is completed in less than 3.5 hours. As this assay is
performed in a 96-well plate, a large number of samples can be handled simultaneously, allowing
for high-throughput automation. This assay is specific for STAT activation and has been shown to
be 10-fold more sensitive and 40-fold faster than the gel retardation technique. With the 3.5-hourprocedure of TransAM, we could detect STAT activation with as little as 0.6 µg of nuclear extract.
A comparable assay using EMSA required 5 µg of nuclear extract and a 5-day autoradiography.
TransAM has many applications including the study of drug potency, inhibitor or activator proteins, and/or protein structure/function in the STAT signaling pathway.

Kit Performance and Benefits
Detection limit: < 0.6 µg nuclear extract/well. The TransAM STAT Family Kit is up to 10-fold moresensitive than EMSA.
Range of detection: TransAM provides quantitative results from 0.3 to 10 µg of nuclear extract
per well.
Cross-reactivity: TransAM STAT Family detects STAT 1α, 3, 5A and 5B from human and rat origin.
STAT3 can also be detected in mouse. Reactivity with other species has not been determined.

STAT activation (OD450 nm)

Assay time: 3.5 hours.

10.75

Extract
Extract with wild-typecompetitor oligos

0.5Extract with mutant
competitor oligos

0.25
0STAT1αSTAT3
STAT5ASTAT family members

STAT5B

Monitoring STAT Family member activation using the TransAM STAT Family Kit. STAT 1α, 3, 5A and 5B activation wereassayed using the TransAM STAT Family Kit. 1:1000 dilutions of STAT1 and STAT5 antibodies and a 1:1000 dilution of STAT3
antibody were tested using 5 µg/well of nuclear extract prepared from a stimulated cell line: STAT1α was tested with COS-7(IFNγ), STAT3 with Hep G2 (IL-6, 100 ng/ml), and STAT5A and STAT5B with Nb2 (prolactin). Assays were performed in theabsence or presence of 20 pmol of competitor oligonucleotide that contains either a wild-type or mutated STAT consensus
binding site. Note that the wild-type oligonucleotide reduces STAT binding by over 90%, while incubation with the mutant
STAT competitor oligo has a limited effect on STAT binding to DNA. This data is provided for demonstration purposes 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.
Reagents

Quantity

Storage / Stability

STAT1α antibody

11 µl-20°C for 6 months

STAT3 antibody

20 µl-20°C for 6 months

STAT5A antibody

11 µl-20°C for 6 months

STAT5B antibody

11 µl-20°C for 6 months

Anti-rabbit HRP-conjugated IgG

2 x 11 µl (0.2 µg/µl)

4°C for 6 months

Wild-type oligonucleotide AM6

100 µl (10 pmol/µl)-20°C for 6 months

Mutated oligonucleotide AM6

100 µl (10 pmol/µl)-20°C for 6 months

Nb2 nuclear extract (prolactin stimulated)

40 µl (2.5 µg/µl)-80°C for 6 monthDithiothreitol (DTT)

100 µl (1 M)-20°C for 6 months

Herring Sperm DNA

100 µl (1 µg/µl)-20°C for 6 months

Protease Inhibitor Cocktail

100 µl

-20°C for 6 months

Lysis Buffer AM1

10 ml

4°C for 6 months

Binding Buffer AM6

10 ml

4°C for 6 months

10X Wash Buffer AM2

60 ml

4°C for 6 months

10X Antibody Binding Buffer AM2

2 x 2.2 ml

4°C for 6 months

Developing Solution

2 x 11 ml

4°C for 6 months

Stop Solution

60 ml

4°C for 6 months

96-well STAT assay plate2

4°C for 6 months

Plate sealer2

Additional materials required• Multi-channel pipettor• Multi-channel pipettor reservoirs• Rocking platform• Microplate spectrophotometer capable of reading at 450 nm (655 nm as optional reference
wavelength)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 cell 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 2 µl of 1 M DTT
and 10 µl of Herring Sperm DNA per ml of Binding Buffer AM6 (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
Washing 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 mayform clumps, 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 STAT3 antibody to 1:1000
and STAT1, STAT5A and STAT5B antibodies and secondary HRP 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.
Nb2 (prolactin stimulated) Nuclear Extract
Nb2 (prolactin stimulated) nuclear extract is provided as a positive control to ensure that the
kit reagents are functional. The Nb2 (prolactin stimulated) extract gives a strong signal for both
STAT5A and STAT5B. This extract is optimized to give a strong signal when used at 5 µg/well.
Sufficient extract is supplied for 20 reactions each. We recommend aliquoting the extract in 5 µl
fractions and storing at -80ºC to help avoid multiple freeze/thaw cycles of the extract.
In addition, positive control nuclear extract for STAT1α and STAT3 can be purchased separately.
COS-7 (IFNγ stimulated) nuclear extract is recommended for STAT1 and Hep G2 (IL-6 stimulated,
100 ng/ml) nuclear extract is recommended for STAT3 (see Appendix, Section B. Related Products).
Wild-type and mutated consensus oligonucleotides
The wild-type consensus oligonucleotide is provided as a competitor for STAT binding in order tomonitor the specificity of the assay. Used at 20 pmol/well, the oligonucleotide will prevent STAT
binding to the probe immobilized on the plate. Conversely, the mutated consensus oligonucleotide should have no effect on STAT binding. Prepare the required amount of wild-type and/or
mutated consensus oligonucleotide by adding 2 µl of appropriate oligonucleotide to 31.8 µl ofComplete 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 cell 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

0.02 µl0.23 µl22.25 µl22.5 µl

0.2 µl
1.8 µl
178.0 µl180.0 µl

1.2 µl10.8 µl
1.068 ml
1.08 ml

2.4 µl21.6 µl2.136 ml
2.16 ml

Complete Binding Buffer

DTT
Herring Sperm DNA
Binding Buffer
Total Required

0.07 µl0.34 µl33.39 µl33.8 µl0.54 µl
2.7 µl266.8 µl
270 µl3.2 µl16.2 µl
1.6 ml1.62 ml

6.5 µl32.4 µ3.2 ml3.24 ml

Binding Buffer with
STAT wt or
mut oligont

wt or mut oligont
Complete Binding Buffer
Total Required

4 µl
41 µl
45.0 µl

32 µl
328 µl360.0 µl

192 µl1.97 ml
2.16 ml

N/A
N/A
N/A

1X Washing Buffer

Distilled Water
10X Washing Buffer
Total Required

2.025 ml
225.0 µl2.25 ml

16.2 ml
1.8 ml18.0 ml97.2 ml
10.8 ml
108.0 ml

194.4 ml21.6 ml
216.0 ml

1X Antibody
Binding Buffer*

Distilled Water
202.5 µl
10X Antibody Binding Buffer 22.5 µl
Total Required
225.0 µl

1.62 ml
180.0 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 µl900.0 µl

5.4 ml10.8 ml

Stop Solution

Total Required

112.5 µl900.0 µl

5.4 ml10.8 ml* Volumes listed refer to the preparation of buffer for diluting both the primary & secondary
antibodies.

STAT Transcription Factor Assay
Determine the appropriate number of microwell strips required for testing samples, controls andblanks in duplicate. If less than 8 wells in a strip need to be used, cover the unused wells witha portion of the plate sealer while you perform the assay. The content of these wells is stable atroom temperature if kept dry and, therefore, can be used later for a separate assay. Store theunused strips in the aluminum pouch at 4°C. Use the strip holder for the assay.
Prepare the Complete Lysis Buffer, Complete Binding Buffer, 1X Wash Buffer and 1X Antibody
Binding Buffer as described above in the section Buffer Preparation and Recommendations.
Multi-channel pipettor reservoirs may be used for dispensing the Complete Binding Buffer, Wash
Buffer, Antibody Binding Buffer, Developing Solution and Stop Solution into the wells being used.

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9

Step 1: Binding of STAT to its Consensus Sequence1.

Add 30 µl Complete Binding Buffer to each well to be used. If you wish to perform competitive binding experiments, add 30 µl Complete Binding Buffer that contains 20 pmol (2 µl)of the wild-type or mutated oligonucleotide (see the Buffer Preparation section above for a
description of competitive binding).

2.

Sample wells: Add 20 µl of sample diluted in Complete Lysis Buffer per well. We recommend using 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 positive control nuclear extract diluted in 20 µl ofComplete Lysis Buffer per well (2 µl of extract in 18 µl of Complete Lysis Buffer per well).
Please refer to page 8 for recommended positive controls for STAT 1α, 3, 5A and 5B.
Blank wells: Add 20 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 of one of the diluted STAT antibodies (1:1000 dilution for STAT1, STAT3, STAT5A
and STAT5B in 1X Antibody Binding Buffer) to each well 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.
16.
17.
18.

Darnell J.E., Kerr I.M. and Stark G.R. (1994) Science 264: 1415-1421.
Zhong Z., Wen Z. and Darnell J.E. (1994) Proc. Natl. Acad. Sci. USA 91: 4806.
Zhong Z., Wen Z. and Darnell J.E. (1994) Science 264: 95.
Ihle J.N. and Kerr I.M. (1995) Trends Genetics 11: 69.
Pallard C. et al. (1995) EMBO J. 14:2847.
Azam M. et al. (1995) EMBO J. 14:1402.
Lin J.X. et al. (1995) Immunity 2: 331.
Quelle F.W. et al. (1995) Mol. Cell. Biol. 15: 3336.
Shuai K., Horvath C.M., Huang L.H. et al. (1994) Cell 76:821-828
Leaman D.W., Leung S., Li X. and Stark G.R. (1996) FASEB J. 10: 1578.
Ihle J.N., Witthuhn B.A., Quelle et al. (1995) Annu. Rev. Immunol. 13: 369.
Schindle, C. and Darnell Jr. J.E., (1995) Annu. Rev. Biochem. 64: 621.
Kim T.K. and Maniatis T. (1996) Science 273: 1717-1719.
Starr R., Willson T.A. and Viney E.M. (1997) Nature 387: 917-921.
Endo T.A., Masuhara M., Yokouchi M. et al. (1997) Nature 386: 921-924.
Naka T., Narazaki M., Hirata M. et al. (1997) Nature 383: 924-929.
Chung C.D., Liao J., Lui B. et al. (1997) Science 278: 1803-1805.
Liu B., Liao J. and Rao X. (1998) Proc. Natl. Acad. Sci. USA 95: 10626-10631.

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.

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.
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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.

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

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

1.5 M NaCl

21.9 g

27 mM KCl

0.5 g

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

For 10 ml, mix:
52 mg

250 mM β-glycerophosphate

0.55 g

250 mM p-nitrophenyl phosphate (PNPP)

1.15 g

25 mM NaVO3

31 mg

Adjust to 10 ml with distilled water. Mix the chemicals by vortexing. Incubate the solution at 50ºC
for 5 minutes. Mix again. Store at –20°C.
PBS/PIB
Prior to use, add 0.5 ml PIB to 10 ml 1X PBS.
HB (Hypotonic Buffer)

For 50 ml, mix:

20 mM Hepes, pH 7.5

0.24 g

5 mM NaF

12 mg

0 µM Na2MoO4

5 µl of a 0.1 M solution

0.1 mM EDTA

10 µl of a 0.5 M solution

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

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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

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 1:1000 for primary antibody and 1:5000
for the secondary antibody. The sensitivity of the assay will bedecreased

Not enough nuclear
extract per well

Increase amount of nuclear extract to 50 µg/well

STAT is poorly activated or inactivated innuclear fractions

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

High background in
all wells

Uneven color
development

High background insample wells

No signal or weak signal
in sample wells

Nuclear extracts
are not from correct
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13

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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14