TransAM™

NFκB p65 Chemi/ NFκB p50 Chemi/
NFκB p52 Chemi(version E3)
Catalog Nos. 40097 & 40597 (NFκB p65)
Catalog Nos. 41097 & 41597 (NFκB p50)
Catalog Nos. 48197 & 48697 (NFκB p52)

Copyright 2014 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.
© 2009 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
NFκB Transcription Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Transcription Factor Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
TransAM NFκB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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
NFκB Transcription Factor Assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Appendix
Section A. Preparation of Nuclear Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section B. Troubleshooting Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

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Overview
The transcription factor NFκB (nuclear factor κB) is a key component for the inducible expressionof a wide variety of cellular and viral genes. Therefore, accurate monitoring of NFκB activation incells, tissues or animals is crucial for drug development and signal transduction pathway studies.
To date, such research projects are time consuming, tedious and lack high-throughput screening
systems.
With its patented TransAM™ method*, Active Motif introduced the first ELISA-based kits to detectand quantify transcription factor activation. TransAM Kits combine a fast, user-friendly format witha sensitive, specific assay. TransAM NFκB p65, p52 and p50 Chemi Kits are designed specificallyfor the study of NFκB subunits. They contain a 96-well plate to which oligonucleotide containing
an NFκB consensus binding site has been immobilized. The activated NFκB homodimers and heterodimers contained in nuclear or whole-cell extracts specifically binds to thisoligonucleotide. By using an antibody that is directed against either the NFκB p65, p52 or p50
subunit, the activated NFκB subunit bound to the oligonucleotide is detected. Addition of a secondary antibody conjugated to horseradish peroxidase (HRP) provides sensitive chemiluminescent readout that is easily quantified using a luminescence measurement. The 96-well plate withdetachable wells is suitable for manual use or high-throughput screening applications. TransAM
NFκB p65, p52 and NFκB p50 Chemi Kits are available in two sizes:

product

formatcatalog no.

TransAM™ NFκB p65 Chemi

1 x 96 rxns
5 x 96 rxns

40097
40597

TransAM™ NFκB p50 Chemi

1 x 96 rxns
5 x 96 rxns

41097
41597

TransAM™ NFκB p52 Chemi

1 x 96 rxns
5 x 96 rxns

48197
48697

The procedure outlined in this manual can be used for either TransAM NFκB p65, TransAM NFκB
p50 or TransAM NFκB p52 Chemi Kits.

* Technology covered by AAT-filed patents and licensed to Active Motif. Use of TransAM in NFκB-related drug discovery may
be covered under U.S. Patent No. 6,150,090 and require a license from Ariad Pharmaceuticals (Cambridge, MA).

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1Flow Chart of Process
3

2

1

5

4

7

6

8

9

10

11

12

A
B
C
D
E

Cell extractcontaining activatedtranscription factor

F
G
H

Oligonucleotide
coated plate

Add cell
extract
21

11

01

9

8

7

6

5

4

3

2

1
A
B

C
D
E
F
G
H

Add primary
antibody

1 hr.
4

3

2

1

9

8

7

6

5

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.
2

1

5

4

3

7

6

A
B
C
D
E
F
G
H

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2

8

9

10

11

12

Introduction
NFκB Transcription Factor
The transcription factor NFκB is implicated in the regulation of many genes that code for mediators of the immune, acute phase and inflammatory responses1. The DNA-binding protein complex
recognizes a discrete nucleotide sequence (5´-GGGACTTTCC-3´) in the upstream region of avariety of cellular and viral response genes2. NFκB is composed of homo- and heterodimericcomplexes of members of the Rel (NFκB) family. There are five subunits of the NFκB family inmammals: p50, p65 (RelA), c-Rel, p52 and RelB3. These proteins share a conserved 300 aminoacid sequence in the N-terminal region, known as the Rel homology domain, that mediates DNA
binding, protein dimerization and nuclear localization. This domain is also a target of the IκB
inhibitors, which include IκBα, IκBβ, IκBγ, Bcl-3, p105 and p1004. Various dimer combinations of
the NFκB subunits have distinct DNA binding specificities and may serve to activate specific setsof genes such as adhesion molecules, immunoreceptors and cytokines. The p50/p65 (NFκB/RelA)
heterodimers and the p50 homodimers are the most common dimers found in the NFκB signaling
pathway. In the majority of cells, NFκB exists in an inactive form in the cytoplasm, bound to theinhibitory IκB proteins5. Treatment of cells with various inducers results in the phosphorylation,ubiquitination and subsequent degradation of IκB proteins6 (For studying the phosphorylation
state of IκBα, see Active Motif’s FunctionELISA™ IκBα Kit). Proteolytic cleavage of p105 results in
two proteins: p50, which has DNA-binding activity but no transactivation domain, and its antagonist, the inhibitory IκBγ protein. This results in the release of NFκB dimers, which subsequently
translocate to the nucleus, where they activate appropriate target genes. NFκB can be activatedby a number of stimuli, including components of bacterial cell walls, such as lipopolysaccharide,or inflammatory cytokines, such as TNF-α or IL-1β.
Transcription Factor Assays
To date, three methods are widely used to measure NFκB activation, either directly or indirectly:
1.

NFκB expression or cytoplasmic IκB degradation can be measured by Western blot, using
antibodies raised against NFκB subunits or IκB (see Appendix, Section C for related products). This method is time consuming (up to 2 days once the cell extracts are prepared), andis not suitable for processing large numbers of samples.

2.

The DNA-binding capacity of NFκB can be assayed by gel retardation, also called electrophoretic mobility shift assay (EMSA). In this method, cell extracts are incubated with aradioactive double-stranded oligonucleotide probe containing the consensus sequence for
NFκB binding. If NFκB is active in the cell 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 NFκB activation is based on reporter genes, typicallyluciferase or β-galactosidase, placed under the control of a promoter containing the NFκB
consensus sequence. This promoter can be artificial, made of several NFκB cis-elements
and a TATA box, or natural, like the HIV long terminal repeat (LTR) sequence. Limitations ofwww.activemotif.com

3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 NFκB
NFκB is a key factor in various immune processes and represents an excellent pharmacologicaltarget. However, pharmaceutical research in this field has been hampered by the lack of convenient assays suitable for large numbers of samples.
To overcome this, Active Motif offers a high-throughput assay to quantify NFκB activation7. The
TransAM Kit combines a fast and user-friendly ELISA format with a sensitive and specific assay
for transcription factors. TransAM NFκB Kits contain a 96-well plate to which an oligonucleotide
containing the NFκB consensus site (5´-GGGACTTTCC-3´) has been immobilized. The active formof NFκB contained in nuclear or whole-cell extract specifically binds to this oligonucleotide. The
primary antibodies used to detect NFκB recognize an epitope on p65, p50 or p52 that is accessible only when NFκB is activated and bound to its target DNA. An HRP-conjugated secondary
antibody provides a sensitive chemiluminescent readout that is easily quantified by luminescence. Once the cellular 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 NFκB activation and has
been shown to be 125-fold more sensitive and 40-fold faster than the gel retardation technique.
With the 3.5-hour procedure of TransAM, we could detect NFκB activation with 40 ng of nuclear
extract from Jurkat cells stimulated with TPA and calcium ionophore. A comparable assay using
EMSA required 5 µg of 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 NFκB signaling pathway.

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4Kit Performance and Benefits
TransAM NFκB Kits are for research use only. Not for use in diagnostic procedures.
Detection limit: < 40 ng nuclear extract/well. For whole-cell extract, twice as much input may beneeded. TransAM NFκB Chemi is 125-fold more sensitive than EMSA.
Range of detection: TransAM Chemi Kits provide quantitative results from 0.039 to 2.5 µg of
nuclear or whole-cell extract per well.
Cross-reactivity:
p65

p50

p52

Human

+

+

+

Mouse

+

+

+

Rat

+

–

–

NFκB p50 activation (RLU)

Assay time: 3.5 hours.

1250000
1000000
750000JurkatJurkat (TPA + CI stimulated)

50000025000000.039 0.078 0.156 0.313 0.625
Extract (µg/well)

1.25

2.5

TransAM NFκB p50 Chemi sensitivity and dynamic range.
Nuclear extracts from Jurkat cells (Catalog No. 36014) and Jurkat cells stimulated with TPA and calcium ionophore (Catalog
No. 36013) were assayed from 0.039 to 2.5 µg/well for NFκB p50 activation using the TransAM NFκB p50 Chemi Kit.

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5Kit Components and Storage
TransAM NFκB 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. All components are guaranteed stable for 6 months from date of receipt when stored
properly.
Quantity
1 plate / 5 plates

Reagents

Storage

NFκB p65 or p50 antibody

11 µl / 55 µl-20°C for 6 months

NFκB p52 antibody

11 µl / 55 µl

4°C for 6 months

Anti-rabbit HRP-conjugated antibody

11 µl / 55 µl

4°C for 6 months

Wild-type oligonucleotide AM20

100 µl / 500 µl(10 pmol/µl)-20°C for 6 months

Mutated oligonucleotide AM20

100 µl / 500 µl(10 pmol/µl)-20°C for 6 months

Positive control nuclear extract

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

Dithiothreitol (DTT) (1 M)

100 µl / 500 µl-20°C for 6 months

Protease Inhibitor Cocktail

100 µl / 500 µl-20°C for 6 months

Herring sperm DNA

100 µl / 500 µl(1 µg/µl)-20°C for 6 months

Lysis Buffer AM2

10 ml / 50 ml

4°C for 6 months

Binding Buffer AM3

10 ml / 50 ml

4°C for 6 months

10X Wash Buffer AM2

22 ml / 110 ml

4°C for 6 months

10X Antibody Binding Buffer AM2

2.2 ml / 11 ml

4°C for 6 months

Chemiluminescent Reagent

2 ml / 10 ml

4°C for 6 months

Reaction Buffer

4 ml / 20 ml

4°C for 6 months

96-well NFκB chemi assay plate

1/5

4°C for 6 months

Plate sealer

1/5

Room temperature

Additional materials required• Multi-channel pipettor• Multi-channel pipettor reservoirs• Rocking platform• Nuclear or whole-cell extracts• Microplate luminometer or CCD camera-couple imaging system (e.g. Tecan GENios
microplate reader)www.activemotif.com

6

Protocols
Buffer Preparation and Recommendations
Preparation of Complete Lysis Buffer
We provide an excess of Lysis Buffer AM2 in order to perform the assay AND to prepare customized nuclear extracts. Please refer to the Appendix Section A for a protocol to prepare a nuclear
extract. Our Nuclear Extract Kit can also be purchased separately (Cat. Nos. 40010 & 40410).
When preparing extracts, it is suggested to perform the final lysis step using the Lysis Buffer AM2
provided in the TransAM Kit. All subsequent dilutions should also be performed using Lysis Buffer
AM2 from the TransAM Kit. Prepare the amount of Complete Lysis Buffer required for the assay
by adding 5 µl of 1 M DTT and 10 µl Protease Inhibitor Cocktail per ml of Lysis Buffer AM2 (seethe Quick Chart for Preparing Buffers in this section). Some of the protease inhibitors lose theiractivity after a few hours once diluted. Therefore, we recommend adding protease inhibitors
immediately prior to use. Any remaining Complete Lysis Buffer should be discarded if not used in
the same day.
Preparation of Complete Binding Buffer
Prepare the amount of Complete Binding Buffer AM3 required for the assay by adding 2 μl of
DTT and 10 μl of 1 μg/µl Herring sperm DNA per ml of Binding Buffer AM3 (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 it is necessary to completely resuspend any precipitates by incubating at 50ºC
for 2 minutes and mixing prior to use.
Preparation of 1X Antibody Binding Buffer
Prepare the amount of 1X Antibody Binding Buffer required for the assay as follows: For every
10 ml of 1X Antibody Binding Buffer required, dilute 1 ml 10X Antibody Binding Buffer 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 it is necessary to completely resuspend
the buffer by warming to room temperature and vortexing for 1 minute prior to use.
* Volumes listed refer to the preparation of buffer for diluting both the primary & secondary
antibodies.
Preparation of Antibody Solutions
Dilute the primary antibody with the 1X Antibody Binding Buffer to 1:1,000 for NFκB p65, p50
and p52. For example, dilute 0.44 µl antibody in 440 µl 1X Antibody Binding Buffer for 1 strip (8wells); multiply these values by the number of strips to be used. Dilute the secondary antibody
to 1:10,000 with the 1X Antibody Binding Buffer by performing a 1:500 dilution followed by a
1:20 dilution. For example, dilute 0.8 µl antibody in 400 µl 1X Antibody Binding Buffer. Next, takewww.activemotif.com

7

22 µl of the previous dilution and add it to 418 µl 1X Antibody Binding Buffer. This is sufficient
reagent for 1 strip (8 wells); multiply these values by the number of strips to be used. Dependingon 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.
Preparation of Chemiluminescent Working Solution
The Chemiluminescent Reagent and Reaction Buffer should be warmed to room temperature
before use. These components are light sensitive, therefore, we recommend avoiding direct
exposure to intense light during storage. Prior to use, place the Chemiluminescent Reagent andReaction Buffer at room temperature for at least 1 hour. In a separate container, mix 1 volume ofChemiluminescent Reagent with 2 volumes of Reaction Buffer to prepare the ChemiluminescentWorking Solution (see the Quick Chart for Preparing Buffers in this section). The Chemiluminescent Working Solution is stable for several hours. After the Chemiluminescent Working Solution isaliquoted into the wells, discard the remaining solution.
Nuclear extract
The Jurkat (TPA + CI) nuclear extract is provided as a positive control for NFκB p50 and p65
activation. The Raji nuclear extract is provided as a positive control for NFκB p52 activation. Sufficient extract is supplied for 80 reactions per plate. The positive control extract is optimized togive a strong signal when used at 1.25 µg/well. We recommend aliquoting the extract in 5 µl fractions and storing at -80°C. Avoid multiple freeze/thaw cycles of the extract. Various cell extracts
are available from Active Motif.
Wild-type and mutated consensus oligonucleotides
The wild-type consensus oligonucleotide is provided as a competitor for NFκB binding in order tomonitor the specificity of the assay. This competition assay will confirm that the protein subunits
binding to the plate are specific for the NFκB consensus binding sequence. Used at 20 pmol/well,
the oligonucleotide will prevent NFκB binding to the probe immobilized on the plate. Conversely,
the mutated consensus oligonucleotide should have no effect on NFκB binding. Prepare therequired amount of wild-type and/or mutated consensus oligonucleotide by adding 2 µl of
appropriate oligonucleotide to 31.8 µl of Complete Binding Buffer per well being used (see the
Quick Chart for Preparing Buffers in this section). To allow for optimum competition, add theoligonucleotide to the well first, then add the cell extract. It is not necessary to perform an incubation step of the oligonucleotide in the well prior to addition of the cell extract. The oligonucleotide competition only needs to be performed as a control. It is suggested to test the oligonucleotide competition each new cell type used.

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8Quick Chart for Preparing Buffers
For 1
well

For 1 strip(8 wells)

For 6 strips(48 wells)

For 12 strips(96 wells)

DTT
Protease Inhibitor Cocktail
Lysis Buffer AM2
TOTAL REQUIRED

0.11 µl0.23 µl22.2 µl22.5 µl

0.9 µl
1.8 µl177.3 µl
180 µl5.4 µl10.8 µl
1.064 ml
1.08 ml

10.8 µl21.6 µl2.128 ml
2.16 ml

Complete Binding Buffer

DTT
Herring sperm DNA
Binding Buffer AM3
TOTAL REQUIRED

0.07 µl0.34 µl33.4 µl33.8 µl0.54 µl
2.7 µl267 µl270 µl3.2 µl16.2 µl
1.6 ml1.62 ml

6.5 µl32.4 µl
3.2 ml3.24 ml

Complete Binding Buffer
with NFκB wild-type ormutated oligonucleotide

NFκB wild-type or mutated oligo
Complete Binding Buffer
TOTAL REQUIRED

2 µl
31.8 µl33.8 µl

16 µl
254 µl270 µl96 µl1.524 ml
1.62 ml

N/A
N/A
N/A

1X Wash Buffer

Distilled water
10X Wash Buffer AM2
TOTAL REQUIRED

2.025 ml
225 µl2.25 ml

16.2 ml
1.8 ml18 ml

97.2 ml
10.8 ml
108 ml194.4 ml21.6 ml
216 ml1X Antibody Binding Buffer*

Distilled water
10X Ab Binding Buffer AM2
TOTAL REQUIRED

157.5 µl17.5 µl
175 µl1.26 ml
140 µl
1.4 ml7.56 ml
840 µl8.4 ml15.12 ml1.68 ml
16.8 ml

ChemiluminescentWorking Solution

Chemiluminescent Reagent
Reaction Buffer
TOTAL REQUIRED

18.7 µl37.5 µl56.2 µl

150 µl300 µl450 µl0.9 ml
1.8 ml
2.7 ml

1.8 ml
3.6 ml5.4 mlReagents to prepare

Components

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

NFκB 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 NFκB 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 0.2-2 µg of nuclear extract diluted in Complete Lysis Buffer per well. A protocol
for preparing nuclear extracts is provided on page 11. For whole-cell extracts we recommend using 0.4-4 µg per well.
Positive control wells: Add 1.25 µg of the provided Jurkat nuclear extract (for p65 or p50)
or Raji nuclear extract (for p52) diluted in 20 µl of Complete Lysis Buffer per well (0.5 µl of
nuclear extract in 19.5 µl of Complete Lysis Buffer per well).
Blank wells: Add 20 µl Complete Lysis Buffer only per well.

Reagents

Blank
wells

Positive Control
no competition

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

30 µl
–
–
20 µl
–

30 µl
–
–
–
20 µl

30 µl
–
–
–
20 µl

28 µl
2 µl
–
–
20 µl

28 µl
–
2 µl
–
20 µl

3.

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

4.

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

Step 2: Binding of Primary Antibody
1.

Add 50 µl of diluted NFκB antibody (1:1,000 dilution in 1X Antibody Binding Buffer) to eachwell being used, including blank wells.

2.

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

3.

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

Step 3: Binding of Secondary Antibody
1.

Add 50 µl of diluted HRP-conjugated antibody (1:10,000 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).
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10Step 4: Chemiluminescent Detection1.

Add 50 µl room-temperature Chemiluminescent Working Solution to all wells being used.
Minimize exposure of the plate to light. Pop any bubbles that remain in the wells to ensure
accurate measurements and then read the plate immediately.

2.

Read chemiluminescence using a luminometer or CCD camera system. You may need tocustomize the plate geometry to work with your plate reader.
Plate length = 127,890 µm

Plate height = 15,000 µm

Plate width = 85,500 µm

Height tolerance = 500 µm

References
1.
2.
3.
4.
5.
6.
7.

Bauerle P.A. and Baichwal V.R. (1997) Adv. Immunol. 65: 111-137.
Parry G.C. and Mackman N. (1994) J. Biol. Chem. 269: 20823-20825.
Verma I.M., Stevenson J.K., Schwarz E.M., Van Antwerp D. and Miyamoto S. (1995) Genes &Dev. 9: 2723-2735.
Gilmore T.D. and Morin P.J. (1993) Trends Genetics 9: 427-433.
Bauerle P.A. and Baltimore D. (1988) Cell 53: 211-217.
Chen Z., Hagler J., Palombella V.J., Melandri F., Scherer D., Ballard D. and Maniatis T. (1995)
Genes & Dev. 9: 1586-1597.
Renard P., Ernest I., Houbion A., Art M., Le Calvez H., Raes M. and Remacle J. (2001) Nucleic
Acids Res. 29:(4), e21.

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11

Appendix
Section A. Preparation of Nuclear Extract
For your convenience, Active Motif offers a Nuclear Extract Kit (Cat. Nos. 40010 & 40410). This
kit contains buffers optimized for use in TransAM Kits, which serves to reduce inconsistencies in
the assay that may arise from using homemade or other buffers. If you prefer to make your own
buffers, please refer to the following protocol.
This procedure can be used for a confluent cell layer of 75 cm2 (100-mm dish). The yield is approximately 0.15 mg of nuclear proteins for 9 x 106 cells.
1.

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

2.

Add 10 ml ice-cold PBS/PIB and scrape the cells off the dish with a cell lifter. Transfer cells
into a pre-chilled 15 ml tube and spin at 300 x g for 5 minutes at 4°C. Do not use Trypsin toremove cells as it may alter transcription factor activation states.

3.

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

4.

Allow the cells to swell on ice for 15 minutes.

5.

Add 50 µl 10% Nonidet P-40 (0.5 % final) and vortex the tube vigorously for 10 seconds.
Check the cells under a microscope to monitor the cell lysis. The cell membrane should becompletely lysed, while the nuclear membrane remains intact.

6.

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

7.

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

8.

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

9.

Determine the protein concentration of the extract by using a Bradford-based assay. It is
recommended to perform a 1:50 dilution of your samples for protein determination. A blanksample consisting of Complete Lysis Buffer diluted 1:50 should be run as control. Standard
controls should also be generated in Complete Lysis Buffer diluted 1:50.

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12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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13Section B: Troubleshooting Guide
Problem/question

Possible cause

Recommendation

No signal or weak signal

Omission of key
reagent

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

Substrate or conjugate
is no longer active

Test conjugate and substrate for activity by mixing HRP and
Chemiluminescent Working Solution together

Enzyme inhibitor
present

Sodium azide will inhibit the peroxidase reaction. Follow ourrecommendations to prepare buffers

Plate reader or CCD
camera settings not
optimalVerify the plate reader is set for luminescence measurementmode or check filter settings on the CCD camera

Incorrect assay
temperature

Bring Chemiluminescent Reagent and Reaction Buffer to room
temperature before using

Inadequate volumeof ChemiluminescentWorking Solution

Check to make sure that correct volume is delivered by pipette

Measurement time
too long

Reduce integration time or exposure time on luminometer or
CCD camera

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 0.01-0.05 µg/well

Concentration of antibodies is too high

Perform antibody titration to determine optimal working concentration. Start using 1:2,000 for primary antibody and 1:50,000
for the secondary antibody. The sensitivity of the assay will bedecreased

Not enough nuclear
extract per well

Increase amount of nuclear extract. Not to exceed 40 µg/well

Too many freeze/thaw
cycles of extract

Aliquot extract into 5 µl aliquots and store at -80°C to avoid
multiple freeze/thaws

NFκB is poorly activated or inactivated innuclear fractions

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

Nuclear extracts
are not from correct
species

The p65, p50 and p52 antibodies work in human and mouse
samples. The p65 antibody also cross-reacts with rat species

Salt concentration toohigh in binding reaction

Reduce amount of extract per well or dialyze extract before use

High background in
all wells

Uneven color
development

High background insample wells

No signal or weak signal
in sample wells

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14

Technical Services
If you need assistance at any time, please call or send an e-mail to Active Motif Technical Service
at one of the locations listed below.

Active Motif North America
Toll free:Direct:Fax:
E-mail:Active Motif Europe
UK Free Phone:France Free Phone:
Germany Free Phone:Direct:Fax:
E-mail:877.222.9543
760.431.1263
760.431.1351
tech_service@activemotif.com
0800/169 31 47
0800/90 99 79
0800/181 99 10+32 (0)2 653 0001
+32 (0)2 653 0050
eurotech@activemotif.com

Active Motif Japan
Direct: +81 (0)3 5225 3638
Fax: +81 (0)3 5261 8733
E-mail: japantech@activemotif.com
Active Motif China
Direct: (86)-21-20926090
Cell Phone: 18521362870
E-mail: techchina@activemotif.com

Visit Active Motif online at activemotif.com

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15