TransAM™ Flexi

NFκB p50 / NFκB p65
Transcription Factor Assay Kits
(version A2)
Catalog No. 41098 (p50)
Catalog No. 40098 (p65)

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 warrantyof 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.

TABLE OF CONTENTS

Page

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Flow Chart of Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Introduction
NFκB Transcription Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Transcription Factor Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Kit Performance and Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Protocols
Buffer Preparation and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Design of Test Oligonucleotides and Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Quick Chart for Preparing Buffers and Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
NFκB Flexi Transcription Factor Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Preparation of Nuclear Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Appendix
Section A. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

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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 original TransAM™ method, Active Motif introduced the first ELISA-based kits to detect andquantify transcription factor activation. These kits contain a 96-well plate that is pre-coated witha consensus binding site oligonucleotide for the factor of interest. This enables convenient assaying of transcription factor binding at this consensus site, but does not allow the study of alternate
binding sites. Because of this, Active Motif has developed the TransAM Flexi Kits, which enable
the study of transcription factor binding to any DNA-binding site. The TransAM Flexi NFκB p50 and
p65 Kits are designed specifically to study NFκB binding at alternative sites. During the assay, abiotinylated oligonucleotide or PCR product, which contains the transcription factor-binding site of
choice, is incubated with a nuclear extract that has been treated to activate NFκB. When incubated together, the active form of NFκB contained in the extract binds to the biotinylated probe. Afterincubation, the extract/probe mixture is then added to the provided streptavidin-coated plate.
The biotinylated probe bound by active NFκB protein is immobilized and any inactive, unboundmaterial is washed away. The bound NFκB transcription factor subunits are detected with specificprimary antibody, for either NFκB p50 or p65. Addition of a secondary antibody conjugated tohorseradish peroxidase (HRP) followed by developing and stop solutions provides a sensitive colorimetric readout that is easily quantified by spectrophotometry. The 96-well plate with individual
strips of 8 wells is suitable for manual use or high-throughput screening applications. TransAM
Flexi has many applications including the study of variant transcription factor binding sites, analysis of native promoters, confirmation of chromatin immunoprecipitation results and determinationof isoform-binding affinity.
product

formatcatalog no.

TransAM Flexi NFκB p50

1 x 96-well plate

41098

TransAM Flexi NFκB p65

1 x 96-well plate

40098

The procedure outlined in this manual can be used for either TransAM Flexi NFκB p50 or
TransAM Flexi NFκB p65 Kits.
Active Motif also offers the original TransAM Kits, which contain the consensus binding site for
NFκB bound to the wells of a 96-well plate. These original TransAM Kits are available in bothcolorimetric and chemiluminescent formats. See these and other Active Motif products related to
the NFκB signaling pathway in Appendix, Section B.

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

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2

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 ofa variety 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 theIκB 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 sets of genes such as adhesion molecules, immunoreceptors and cytokines. The p50/
p65 (NFκB1/RelA) heterodimers and the p50 homodimers are the most common dimers foundin NFκB signaling pathway. In the majority of cells, NFκB exists in an inactive form in the cytoplasm, bound to the inhibitory IκB proteins5. Treatment of cells with various inducers results inthe 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 antagonist proteins: p50, which has DNA-binding activity but
no transactivation domain, and 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 activated by 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 B for related products). 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 NFκB can be assayed by gel retardation, also called
electrophoretic mobility shift assay (EMSA). In this method, cell extracts are incubatedwith a radioactive 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 then resolved by electrophoresis on a native polyacrylamide gel, followedby autoradiography. This method is sensitive, but like the previous procedure, it is time
consuming (multiple days of gel exposure may be required to achieve sufficient sensitivity)and it cannot be applied to high-throughput screening. Gelshift assays also require special
precautions and equipment for handling radioactivity.

3.

Another method used to assay 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
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3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.

Kit Performance and Benefits
The TransAM Flexi NFκB Kits are for research use only. Not for use in diagnostic procedures.
Detection limit: > 0.5 µg nuclear or whole-cell extract/well or > 0.4 ng recombinant p50 or p65
protein/well. TransAM Flexi is 10-fold more sensitive than EMSA.
Range of detection: TransAM Flexi provides quantitative results from 0.5 to 10 µg of nuclear orwhole-cell extract/well.
Cross-reactivity: TransAM NFκB specifically detects bound NFκB p65 in human, mouse and rat extracts
and bound p50 in human extracts. The p50 antibody has limited reactivity with rat samples.

NFkB activation (OD450 nm)

Assay time: 3.5 hours. TransAM is 40-fold faster than EMSA.

0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0

Untreated
Treated50 bp
5´

TF Binding site
B

Igk - GGAAAGTCCC
IL-8 - GGAAATTCCA
uPA - GTACTTTCCC

consensus Igk
control

IL-8

uPA

Monitoring NFκB binding affinity to natural binding sites using TransAM NFκB p65 Flexi.
Five µg of nuclear extract from untreated and TNF-α-treated HeLa cells were used to assay for NFκB binding
affinity to four different 50-mer oligonucleotides. Three oligos were synthesized to represent the natural binding sites on various promoters regulated by NFκB7: Igκ (Accession NG000833), IL-8 (Accession M28130) anduPA (Accession X12641). Binding affinity from the natural binding sites was compared with the biotinylatedconsensus binding site control provided in the TransAM Flexi Kit. This data is provided for demonstration only.

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43´

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

NFκB p50 or p65 antibody

11 µl-20°C for 6 months

Anti-rabbit HRP-conjugated IgG

11 µl

4°C for 6 months

Biotinylated oligonucleotide

40 µl(1 pmol/µl)-20°C for 6 months

Wild-type oligonucleotide AM20

100 µl(10 pmol/µl)-20°C for 6 months

Mutated oligonucleotide AM20

100 µl(10 pmol/µl)-20°C for 6 months

Jurkat nuclear extract (TPA + CI stimulated)

40 µl(2.5 µg/µl)-80°C for 6 months

Dithiothreitol (DTT)

100 µl
(1 M)-20°C for 6 months

Protease Inhibitor Cocktail

100 µl

-20°C for 6 months

Herring sperm DNA

100 µl
(1 µg/µl)-20°C for 6 months

Binding Buffer AM3

10 ml

4°C for 6 months

10X Wash Buffer AM2

22 ml

4°C for 6 months

10X Antibody Binding Buffer AM2

2.2 ml

4°C for 6 months

Developing Solution

11 ml

4°C for 6 months

Stop Solution

11 ml

4°C for 6 months

96-well assay plate1

4°C for 6 months

Plate sealer

1

Additional materials required• Multi-channel pipettor

For Nuclear Extract preparation• Multi-channel pipettor reservoirs• Lysis Buffer• Rocking platform• Hypotonic Buffer• 1.5 ml microcentrifuge tubes• Phosphatase Inhibitor Buffer• Microplate spectrophotometer capableof reading at 450 nm (655 nm as
optional reference wavelength)• 10X PBS

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5

Protocols
Buffer Preparation and Recommendations
Preparation of Complete Binding Buffer
Prepare the amount of Complete Binding Buffer required for the assay by adding 1 µl of 1 M DTT,
10 µl Protease Inhibitor Cocktail (PIC) 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 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
to room temperature and vortexing for 1 minute prior to use. Dilute both primary and secondary
antibodies 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.
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.

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6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.

Positive control - Nuclear extract
The Jurkat nuclear extract (TPA + CI stimulated) is provided as a positive control for NFκB activation. Sufficient extract is supplied for 20 reactions per plate. This extract is optimized to give astrong signal when used at 5 µg/well. We recommend aliquoting the extract in 5 µl fractions andstoring at -80ºC. Avoid multiple freeze/thaw cycles of the extract. Various cell extracts are available from Active Motif (see Appendix, Section B. Related Products).
Positive control - Biotinylated oligonucleotide
The biotinylated oligonucleotide is provided as a positive control for NFκB binding and is a 50 bp
oligonucleotide that contains the consensus binding site 5´-GGGACTTTCC-3´ for NFκB. This oligo
is supplied at 1 pmol/µl and 1 pmol per reaction should be used.
Design of test biotinylated oligonucleotides or PCR fragments
The advantage of the TransAM Flexi Kit is that it enables the evaluation of different binding sites
for NFκB within the same kit (see Figure on page 4). For optimal results, probes should be aminimum of 50 bp with a maximum length of 2 kb. Probes can either be synthesized or generated by PCR.
a.

Once the binding site of interest is determined, synthesize two complementary oligos. In
all cases the oligo probes should be biotinylated on one end. Biotinylation can be on either
the 5´ or 3´ end of the probe. It is recommended that the binding site of interest be locateddistal to the biotinylated end. For example, if the oligo is biotinylated on the 5´ end, thebinding site should be located closer to the 3´ end. We recommend leaving a minimum of 5
bp between the binding site and the end of the oligo.

b.

Combine the two complementary oligos at an equimolar ratio and an approximate concentration of 100 µM, in a volume of 100-500 µl.

c.

Heat the oligo mixture at 95°C for 10 minutes in a microcentrifuge tube on a heating blockand allow the block containing the mixture to cool down slowly to room temperature.

d.

After diluting the double-stranded oligo to its desired concentration (we recommend
1 pmol/µl), it is ready to use.

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7

Wild-type and mutated consensus oligonucleotides
Active Motif provides unlabeled wild-type and mutated oligonucleotides to perform competition
experiments against the provided biotinylated oligonucleotide. The wild-type consensus oligonucleotide, when used at 10 pmol/well, will prevent NFκB binding to the probe immobilized on
the plate. Conversely, the mutated consensus oligonucleotide should have no effect on NFκB
binding. Prepare the required amount of wild-type and/or mutated consensus oligonucleotide
by adding 1 µl of appropriate oligonucleotide to the appropriate sample per well being used. To
allow for optimum competition, add the oligonucleotide to the Complete Binding Buffer prior toaddition of the cell extract.
If you have synthesized a biotinylated probe for use with TransAM Flexi and would like to perform competition experiments, unlabeled wild-type and mutated competitor oligonucleotides
should also be prepared. As a general rule, it is best to limit the number of nucleotides that arealtered in the mutated oligo to no more than 4 within a given DNA binding site.
OPTIONAL – Preparation of standard curve
For those who wish to quantify the amount of NFκB in their samples, Active Motif offers recombinant NFκB p50 and p65 for use as protein standards (see Appendix, Section B. Related Products).
1.

Begin with a 100 ng/µl working stock of recombinant protein (use the TransAM Complete
Binding Buffer to dilute the protein). Set up a standard curve in duplicate using the following
concentrations: 0.5, 0.25, 0.125, 0.0625, 0.0312, 0.0156, 0.008 and 0 ng/µl. Note: The preceding range is provided as guidance, a broader range of values may be used. Please see the
lot specific technical sheet that accompanies each protein for specific standard curve determination.

2.

Make up a 0.5 ng/µl solution by adding 1 µl of the 100 ng/µl working stock to 199 µl ofComplete Binding Buffer. Next, pipette 60 µl of Complete Binding Buffer into the 7 remaining tubes. Use the 0.5 ng/µl solution to prepare a dilution series as indicated below. Be
sure to mix each tube thoroughly before each transfer. The 0.5 ng/µl standard serves as
the high standard, while Complete Binding Buffer alone serves as the 0.0 ng/µl.

60 µl

0.5 ng/µl
3.

60 µl

0.25 ng/µl

60 µl

60 µl

60 µl

60 µl

0.125 ng/µl 0.0625 ng/µl 0.0312 ng/µl 0.0156 ng/µl 0.008 ng/µl

20 µl from each tube will be diluted into the Complete Binding Buffer and Biotinylated probe
used in Step 1, No. 1 of the protocol and will correspond to the following quantities of NFκB:
10, 5, 2.5, 1.25, 0.625, 0.312, 0.156 and 0.0 ng/well.

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8Quick Chart for Preparing Buffers and Samples*
For sample wells, please add components in the order listed below.
Reagents to prepare

Components

For 1
well

For 1 strip(8 wells)

For 6 strips(48 wells)

For 12 strips(96 wells)

Sample Wells
2 µg extract/well

Binding Buffer AM3
DTT
Protease Inhibitors
Herring sperm DNA
Biotinylated oligo (1 pmol/µl)
2 µg Extract at 2.5 µg/µl
TOTAL REQUIRED

51.965 µl
0.055 µl0.55 µl0.55 µl
1.0 µl0.88 µl55 µl424.35 µl
0.45 µl
4.5 µl
4.5 µl9.0 µl7.2 µl450 µl

2.452 ml
2.6 µl
26 µl
26 µl
52 µl
41.6 µl
2.6 ml4.904 ml
5.2 µl52 µl
52 µl
104 µl83.2 µl
5.2 mlSample Wells
5 µg extract/well

Binding Buffer AM3
DTT
Protease Inhibitors
Herring sperm DNA
Biotinylated oligo (1 pmol/µl)
5 µg Extract at 2.5 µg/µl
TOTAL REQUIRED

50.645 µl
0.055 µl0.55 µl0.55 µl
1.0 µl2.2 µl55 µl413.55 µl
0.45 µl
4.5 µl
4.5 µl9.0 µl18 µl450 µl2.389 ml
2.6 µl
26 µl
26 µl
52 µl
104 µl
2.6 ml4.779 ml
5.2 µl52 µl
52 µl
104 µl
208 µl5.2 mlSample Wells
10 µg extract/well

Binding Buffer AM3
DTT
Protease Inhibitors
Herring sperm DNA
Biotinylated oligo (1 pmol/µl)
10 µg Extract at 2.5 µg/µl
TOTAL REQUIRED

48.445 µl
0.055 µl0.55 µl0.55 µl
1.0 µl
4.4 µl55 µl395.55 µl
0.45 µl
4.5 µl
4.5 µl9.0 µl36 µl450 µl2.285 ml
2.6 µl
26 µl
26 µl
52 µl
208 µl
2.6 ml4.571 ml
5.2 µl52 µl
52 µl
104 µl416 µl5.2 mlSample Wells
20 µg extract/well

Binding Buffer AM3
DTT
Protease Inhibitors
Herring sperm DNA
Biotinylated oligo (1 pmol/µl)
20 µg Extract at 2.5 µg/µl
TOTAL REQUIRED

44.045 µl
0.055 µl0.55 µl0.55 µl
1.0 µl
8.8 µl55 µl359.55 µl
0.45 µl
4.5 µl
4.5 µl9.0 µl72 µl
450 µl

2.077 ml
2.6 µl
26 µl
26 µl
52 µl
416 µl
2.6 ml4.155 ml
5.2 µl52 µl
52 µl
104 µl832 µl5.2 ml

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

93.6 ml
10.4 ml
104 ml187.2 ml
20.8 ml
208 ml1X Antibody
Binding Buffer**

Distilled water
10X Ab Binding Buffer AM2
TOTAL REQUIRED

202.5 µl22.5 µl
225 µl1.62 ml
180 µl
1.8 ml9.36 ml1.04 ml
10.4 ml18.72 ml
2.08 ml
20.8 ml

Developing Solution

TOTAL REQUIRED

112.5 µl

900 µl5.2 ml10.4 ml

Stop Solution

TOTAL REQUIRED

112.5 µl

900 µl5.2 ml10.4 ml* Volumes listed in the Quick Chart contain an excess of components to perform the assay.
** Volumes listed for the 1X Ab Binding Buffer refer to the preparation of buffer for diluting boththe primary & secondary antibodies.

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9

NFκB Flexi 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 Binding Buffer, 1X Wash Buffer and 1X Antibody Binding Buffer as describedin the section Buffer Preparation and Recommendations. Multi-channel pipettor reservoirs may
be used for dispensing the Wash Buffer, Antibody Binding Buffer, Developing Solution and Stop
Solution.
Step 1: Binding of NFκB to the biotinylated probe1.

All samples should be diluted to 50 µl with Complete Binding Buffer in microcentrifuge
tubes. Multiple repeats of the same binding assay can be multiplexed and mixed in the same
tube. For best results, the nuclear extract should be added to the binding reaction mixture
last. Please refer to the Quick Chart on page 9 for directions on preparing sample wells.
Sample wells: Prepare sample by adding 1 pmol of biotinylated probe and the desired amount of
nuclear extract diluted to 50 µl in Complete Binding Buffer. We recommend using 2-20 µg (at 2.5µg/µl) of nuclear extract per well. A protocol for preparing nuclear extracts is provided on page 12.
Positive control wells: Add 1 pmol (1 µl) of the provided biotinylated oligonucleotide and 5 µg
(2 µl) of the provided Jurkat nuclear extract diluted to 50 µl in Complete Binding Buffer.
Blank wells: Add 1 pmol biotinylated probe diluted to 50 µl in Complete Binding Buffer only.
OPTIONAL – Competitive binding wells: If you wish to perform competitive binding experiments, add 10 pmol of the appropriate wild-type or mutated oligo to each sample being used(see page 8 for a description of competitive binding). If using the competition oligos provided in the kit, add 1 µl (10 pmol) wild-type or mutated oligo to each sample being used.
OPTIONAL – Protein standard wells: Add 1 pmol of the biotinylated probe and 20 µl of
the appropriate protein standard diluted to 50 µl in Complete Binding Buffer (see page 8,
Preparation of standard curve).

2.

Mix each tube from above by vortexing and then incubating at room temperature for 30 minutes.

3.

Transfer the entire 50 µl reaction to an individual well on the plate. Use the provided adhesive cover to seal the plate. Incubate for 1 hour at room temperature with mild agitation.

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 NFκB 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.

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

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10Step 3: Binding of secondary antibody1.

Add 100 µl diluted HRP 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. Please read theCertificate of Analysis supplied with this kit for optimal development time for this specific lot.
Monitor the blue color development in the sample 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 optionalreference wavelength of 655 nm. Blank the plate reader according to the manufacturer’s
instructions using the blank wells.

OPTIONAL – Calculation of results using the standard curve
If you have generated a standard curve using Active Motif’s recombinant NFκB p50 or p65 protein,
average the duplicate readings for each standard, control, and sample and subtract the opticaldensity (OD) obtained from the zero standard. Plot the OD for the standards against the quantity (ng/well) of the standards and draw the best fit curve. The data can be linearized using log/log paper andregression analysis may also be applied. To quantify the amount of NFκB in the samples, find theabsorbance value for the samples on the y-axis and extend a horizontal line to the standard curve.
At the intersection point extend a vertical line to the x-axis and read the corresponding standard
value. Note: If the samples have been diluted, the value read from the standard curve must be multiplied by the dilution factor. The following standard curve is provided for demonstration only.
NFkB p50 TransAM Assayy = 0.468x - 0.055 r = 0.999

1.5

A450

1
A450

0.5

0

0

0.5

1

1.5

2

2.5

p50 Recombinant Protein (ng)www.activemotif.com

113

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 the 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 0.5 mg of nuclear proteins for 107 cells.
1.

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

2.

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

3.

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

4.

Allow the cells to swell on ice for 15 minutes.

5.

Add 50 µl 10% Nonidet P-40 (0.5 % final) and mix by gentle pipetting.

6.

Centrifuge the homogenate for 30 seconds at 4°C in a microcentrifuge.

7.

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

8.

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

9.

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

10X PBS
0.1 M phosphate buffer, pH 7.5
1.5 M NaCl
27 mM KCl

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

Adjust to 250 ml with distilled water. Prepare a 1X PBS solution by adding 10 ml 10X PBS to 90
ml distilled water. Sterilize the 1X PBS by filtering through a 0.2 µm filter. The 1X PBS is at pH 7.5.
Store the filter-sterilized 1X PBS solution at 4°C.
PIB (Phosphatase Inhibitor Buffer)
125 mM NaF
250 mM β-glycerophosphate
250 mM para-nitrophenyl phosphate (PNPP)
25 mM NaVO3

For 10 ml, mix
52 mg
0.55 g1.15 g
31 mg

Adjust to 10 ml with distilled water. Mix the chemicals by vortexing. Incubate the solution at 50ºC
for 5 minutes. Mix again. Store at –20°C.

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12PBS/PIB
Prior to use, add 0.5 ml of PIB to 10 ml of 1X PBS.
HB (Hypotonic Buffer)
20 mM Hepes, pH 7.5
5 mM NaF
10 µM Na2MoO4
0.1 mM EDTA

For 50 ml, mix
0.24 g
12 mg
5 µl of a 0.1 M solution
10 µl of a 0.5 M solution

Adjust pH to 7.5 with 1 N NaOH. Adjust volume to 50 ml with distilled water. Sterilize by filtering
through a 0.2 µm filter. Store the filter-sterilized solution at 4°C.
Lysis Buffer
20 mM Hepes, pH 7.5
400 mM NaCl
0.1 mM EDTA
10 mM NaF
10 µM Na2MoO4
1 mM NaVO3
20% glycerol
10 mM PNPP
10 mM beta-glycerophosphate

For 50 ml, mix:
0.24 g1.17 g
1.5 mg
21 mg
0.12 mg
6.1 mg
10 ml
0.23 g0.11 gAdjust pH to 7.5 with 1 N NaOH. Adjust volume to 50 ml with distilled water. Store at 4°C. Just
before use, make up Complete Lysis Buffer by adding 1 µl of 1 M DTT and 10 µl of Protease
Inhibitor Cocktail (Sigma, Cat. No. P8340) per ml of Lysis Buffer.

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.
Parry G. and Mackman N. (1994) J. Biol. Chem. 269: 20823-20825.

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13

Appendix
Section A. Troubleshooting Guide
PROBLEMPOSSIBLE CAUSE

RECOMMENDATION

No signal or weak signal
in all wells

Omission of key reagent

Check that all reagents have been addedin the correct order

Substrate or conjugate is nolonger active

Test conjugate and substrate for activity

Enzyme inhibitor present

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

Plate reader settings not optimal

Verify the wavelength and filter settings in
the plate reader

Incorrect assay temperature

Bring substrate to room temperature

Inadequate volume ofDeveloping Solution

Check to make sure that correct volume
is delivered by pipette

Developing time too long

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

Concentration of antibodies
too high

Increase antibody dilutions

Inadequate washing

Ensure all wells are filled with Wash Buffer
and follow washing recommendations

Incomplete washing of wells

Ensure all wells are filled with Wash Buffer
and follow washing recommendations

Well cross-contamination

Follow washing recommendations

Too much cellular extract per well

Decrease amount of cellular extract down to
1-2 µg/well

Concentration of antibodies
too high

Perform antibody titration to determine
optimal working concentration. Start using
1:2000 for primary antibody and 1:5000 for thesecondary antibody. The sensitivity of theassay will be decreased

Not enough cellular extract per well

Increase amount of cellular extract not toexceed 50 µg/well

Using a whole-cell extract

Prepare a nuclear extract according to theprotocolNFκB is poorly activated orinactivated

Perform a time course for NFκB activationin the studied cell line

Extracts are not fromcorrect origin

The p65 antibody in TransAM NFκB p65
detects human, mouse and rat p65. The p50
antibody in TransAM NFκB p50 is weakly
reactive with rodent p50. You may need to a
add double or triple the amount of recom-

High background in all wells

Uneven color development

High background in sample wells

No signal or weak signal insample wells

mended extract to obtain a signal.

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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

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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