TransAM™ IRF-3 (Human)(version A3)
Catalog Nos. 48396 & 48896

Copyright 2008 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.
© 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
IRF Transcription Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Transcription Factor Assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
TransAM IRF-3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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
IRF-3 (Human) 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
Interferon Regulatory Factors (IRFs) constitute a family of transcription factors that are involvedin host defense via regulation of anti-viral immune responses, cell growth and hematopoietic
development. Therefore, accurate monitoring of IRF activation in cells, tissues and animals is
crucial for many biomedical research and drug development projects. To date, such research
projects are tedious and time consuming, and lack high-throughput screening methods.
With its patented TransAM™ method*, Active Motif introduced the first ELISA-based kits to detectand quantify transcription factor activation. TransAM Kits combine a fast, user-friendly format
with a sensitive, specific assay. TransAM IRF-3 Kits are designed specifically to detect and quantify IRF-3 activation. They contain a 96-well plate to which oligonucleotide containing an interferon regulatory factor element (IRFE) has been immobilized. IRF-3 contained in nuclear extractbinds specifically to this oligonucleotide and is detected through use of an antibody directedagainst IRF-3. Addition of a secondary antibody conjugated to horseradish peroxidase (HRP) provides a sensitive colorimetric readout that is easily quantified by spectrophotometry. The 96-wellplate with individual strips of 8 wells is suitable for manual use or for high-throughput screening
applications.

product

formatcatalog no.

TransAM™ IRF-3 (Human)

1 x 96 rxns

48396

TransAM™ IRF-3 (Human)

5 x 96 rxns

48896

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

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1Flow Chart of Process

2

1

5

4

3

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.
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
IRF Transcription Factor
The interferon (IFN) regulatory factor (IRF) family is a group of transcription factors that haveextensive homology in their DNA-binding domain (DBD)1. The many members of the IRF family
are involved in the regulation of interferon (IFN) a and b and play a role in host anti-viral immune
regulation, cell growth and hematopoietic development2. The N-terminal binding domain of
IRFs, the distinct feature of the family, is a modified helix-turn-helix characterized by repeatedtryptophan residues separated by 10 to 18 amino acids3. All IRFs, except IRF-1 and IRF-2, have an
IRF association domain (IAD) that is responsible for the interaction with other family members orwith transcription factors such as PU.1, E47 and STAT. Another association domain (IAD2), present
only in IRF-1 and IRF-2, is important for interaction with IRF-8. A nuclear localization signal has
been identified in IRF-1, and similar sequences may also be present in other family members4. A
bipartite nuclear retention signal located within the N-terminus of the DBD has been identified inIRF-4, IRF-8 and IRF-9. IRFs also possess a transactivation domain in the middle of the protein.
IRFs bind DNA as dimers on sequences such as the IFN-stimulated response element (ISRE),
AGTTTCNNCNY, the IFN consensus sequence (ICS), R(G/C)TTTC, or the IFN-regulatory factor element (IRFE), G(A)AAA(G/C)YGAAA(G/C)Y. While IRF-3 is constitutively expressed, the expression ofother IRFs is induced by such stimuli as type I and II IFN, double-stranded RNA or the presenceof viral components, which can also induce the activity of the IRF factors after they have beensynthesized. IRF factors can cooperate with other factors with neighboring binding sites on
promoters. For example, the IFN-β promoter provides the stepping stone for the formation of an“enhanceosome” containing ATF-2, c-Jun, IRF-3, NFκB and CBP.
In mammals, ten IRF family members have been identified5. IRF-1, an activator, is involved inmature lymphocyte apoptosis after DNA-damage. IRF-2 is mainly a repressor, but can also playa role in histone H4 expression. Both IRF-1 and IRF-2 interact with the co-activator P/CAF. IRF-1
has been described as a tumor suppressor and IRF-2 as a proto-oncogene. IRF-3 is a component of DRAF, a complex containing the acetylase CBP/p300, which binds ISRE-like sequences.
Double-stranded RNA (dsRNA) or poly (I-C), a synthetic form of dsRNA, elicits IRF3 activation6.
IRF-4 is required for the function and homeostasis of B and T-cells and can also interact with the
Ets-family member PU.1 and with the E47 form of E2A. IRF-7 helps induce the IFN-α gene andrepress the EBNA-1 gene from the Epstein-Barr virus. The expression of IRF-7 is induced by type
I IFN, which is important for amplification of the signaling pathway7. IRF-8 (ICSBP) can interact
with IRF-1 and 2, but primarily acts as a repressor. IRF-9 (ISGF3γ/p48) is part of the ISGF3 transcription factor, together with STAT1 and STAT2. IRF-10 functions in the late stages of anti-viral
defense by regulating IFNγ target genes.

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3

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

IRF activation can be measured by native Western blot to observe IRF-3 dimers by using
antibodies specific for IRF proteins. This method is time consuming (up to 2 days once
the nuclear extracts are prepared), and is not suitable for processing large numbers ofsamples.

2.

The DNA-binding capacity of IRF 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
IRF binding. If IRF 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
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 IRF activation is based on reporter genes, typically luciferase or β-galactosidase, placed under the control of a promoter containing the IRF consensus sequence. The promoter can be artificial, made of several IRF cis-elements and a TATA
box, or natural, like the GAAANN purine-rich virus response element (VRE) in the IFNA promoter8. However, the procedure is limited by the following issues: (i) reporter gene assays
have to be repeated several times to obtain statistically reliable data; and (ii) reporter 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 sensitive and easy to perform with a large number of samples but requires efficient cell transfection with the reporter plasmid.

TransAM IRF-3
The family of interferon regulatory factor (IRF) transcription factors play an important role in theregulation of interferons in response to infection by virus and in the regulation of interferoninducible genes. As the function and activity of individual IRFs can depend both on their activation state as well as that of other IRF family members, the ability to simultaneously profile theactivation states of different IRF family members is critical to understanding their role in theregulation of immune function and oncogenesis.
To help achieve this, Active Motif offers high-throughput assays to quantify activated IRF-3
and IRF-79. The TransAM Kit combines a fast and user-friendly ELISA format with a sensitive and specific assay for transcription factors. TransAM IRF-3 Kits contain a 96-well plateon which has been immobilized oligonucleotide containing the IRF consensus binding site
(5´-GAAACTGAAACT-3´). The active form of IRF-3 contained in nuclear extract specifically binds
to this oligonucleotide. The primary antibody used to detect IRF recognizes an epitope on IRF-3
protein upon DNA binding. Addition of an HRP-conjugated secondary antibody provides a sensitive colorimetric readout easily quantified by spectrophotometry. Once the nuclear extracts areprepared, this assay is completed in less than 3.5 hours. As this assay is performed in 96-wellplates, a large number of samples can be handled simultaneously, enabling high-throughput
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4automation. This assay is specific for IRF-3 activation and has been shown to be 5-fold moresensitive and 20-fold faster than the gel-retardation technique. With the 3.5-hour procedure ofTransAM, we could detect IRF activation using as little as 0.5 µg of nuclear extract.
TransAM has many applications including the study of drug potency, inhibitor or activator proteins, and/or protein structure/function in the IRF signaling pathway.

Kit Performance and Benefits
Detection limit: < 0.5 µg nuclear extract/well.
Range of detection: TransAM provides quantitative results from 0.5 to 10 µg of nuclear extract
per well.
Cross-reactivity: TransAM IRF-3 specifically detects IRF-3 from human origin. For a TransAM
IRF-3 kit that is specific for mouse and rat, please see catalog numbers 48496 and 48996.
Assay time: 3.5 hours.

IRF-3 activation (OD 450 nm)

1.5
1.2
0.9
0.6
0.3
0.0

0.625

2.5
1.25
5
Nuclear Extract (µg/well)

10

Monitoring IRF-3 activation with the TransAM IRF-3 (Human) Kit:
0.625 to 10 µg of Cos-7 (poly I-C treated, 2 hr) nuclear extract were assayed per well. Data shown are the results from wells
assayed in duplicate.

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5Kit Components and Storage
TransAM IRF-3 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. Avoid repeated freezing and thawing of the IRF-3 antibody. All components are guaranteed stable for 6 months from date of receipt when stored properly.
Reagents

Quantity
1 plate / 5 plates

Storage

IRF-3 antibody (human)

10 µl / 25 µl

4°C

anti-mouse HRP-conjugated antibody

10 µl / 50 µl

4°C

Wild-type oligonucleotide AM6

100 µl / 500 µl(10 pmol/µl)-20°C

Mutated oligonucleotide AM16100 µl / 500 µl(10 pmol/µl)

-20°C

Cos-7
nuclear extract (Poly (I-C) 2 hr)

40 µl / 200 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-20°C

Lysis Buffer AM1

10 ml / 50 ml

4°C

Binding Buffer AM2

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 IRF-3 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 IRF during theextract preparation and the assay. The presence of these inhibitors gives a yellow coloration to
Lysis Buffer AM1. Prepare the amount of Complete Lysis Buffer required for the assay by adding
1 µl of 1 M DTT and 10 µl 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 1 µl of 1 M DTT
and 10 µl of 17 µg/ml poly[d(I-C)] per ml of Binding Buffer AM2 (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 form clumps, therefore homogenize the buffer by incubating at 50ºC for 2 minutes and mixing prior to use.
Preparation of 1X Antibody Binding Buffer
Prepare the amount of 1X Antibody Binding Buffer required for the assay as follows: For every
10 ml of 1X Antibody Binding Buffer required, dilute 1 ml 10X Antibody Binding Buffer 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 1:1000 for the IRF-3 and the HRP-conjugated secondary antibodies.
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 atroom temperature for at least 1 hour. Transfer the amount of Developing Solution required forthe 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 when handling, i.e. safety glasses, gloves and labcoat.
Cos-7 nuclear extract (Poly (I-C) 2 hr)
The Cos-7 (Poly (I-C) 2 hr) nuclear extract is provided as a positive control for IRF-3 activation.
Sufficient extract is supplied for 10 reactions if using 10 µg per well. This extract is optimized togive a strong signal when used at 10 µg/well. We recommend aliquoting the extract in 5 µl fractions and storing at -80ºC. Avoid multiple freeze/thaw cycles of the extract.
Wild-type and mutated consensus oligonucleotides
The wild-type consensus oligonucleotide is provided as a competitor for IRF binding in order tomonitor the specificity of the assay. Used at 20 pmol/well, the oligonucleotide will prevent IRF
binding to the probe immobilized on the plate. Conversely, the mutated consensus oligonucleotide should have no inhibitory effect on IRF binding. Prepare the required amount of wild-type
and/or mutated consensus oligonucleotide by adding 2 µl of appropriate oligonucleotide to 43 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 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 µl1.07 ml
1.08 ml

Complete Binding Buffer

DTT
Poly [d(I-C)]
Binding Buffer
Total Required

0.04 µl0.45 µl44.5 µl
45 µl

0.3 µl
3.6 µl356.1 µl
360 µl2.16 µl21.6 µl2.14 ml
2.16 ml

4.3 µl43.2 µl4.27 ml
4.32 ml

Binding Buffer with
IRF wt or mut oligont

wt or mut oligont
Complete Binding Buffer
Total Required

2.0 µl43.0 µl45.0 µl

16.0 µl344.0 µl360.0 µl96.0 µl
2.064 µl2.16 µl

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
10X Antibody Binding Buffer
Total Required

202.5 µl22.5 µl225.0 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.

IRF-3 (Human) 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 IRF 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 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 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 10 µg of the provided nuclear extract diluted in 10 µl ofComplete Lysis Buffer per well (4 µl of nuclear extract in 6 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 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 IRF-3 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).

1.

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).

1.

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.

Step 3: Binding of secondary antibody

Step 4: Colorimetric reactionwww.activemotif.com

10References
1.
2.
3.
4.
5.
6.
7.
8.
9.

Mamane Y., et al. (1999) Gene 237(1): 1-14.
Harada H., et al. (1998) Biochimie 80(8-9): 641-50.
Nguyen H., et al. (1997) Cytokine Growth Factor Rev. 8(4): 293-312.
Schaper F., et al. (1998) Biochem J. 335 (Pt 1): 147-57.
Taniguchi T., et al. (2001) Annu. Rev. Immunol. 19: 623-655.
Guofeng C. et al. (2007) PNAS 104:9035-9040.
Hiscott J. (2007) J Biol. Chem. 282:15325-15329.
Au W.C. and Pitha P.M. (2001) J. Biol. Chem. 276: 41629-41637.
Renard P., et al. (2001) Nucleic Acids Res. 29: (4) e21.

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 isapproximately 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
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 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 PBSFor 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.
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

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

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

IRF is poorly activated or inactivated innuclear fractions

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

High background in all
wells

Uneven color development

High background in sample
wells

No signal or weak signal insample wells

Nuclear extracts
are not from correct
specieswww.activemotif.com

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