TransAM™
NFkB p65 / NFkB p50 /NFkB p52
Transcription Factor Assay Kits
(version G1)
Catalog Nos. 40096 & 40596 (p65)
Catalog Nos. 41096 & 41596 (p50)
Catalog Nos. 48196 & 48696 (p52)
Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, California 92008, USA
Toll free:
877 222 9543
Telephone:
760 431 1263
Fax:
760 431 1351
Active Motif Europe
104 Avenue Franklin Roosevelt
B-1330 Rixensart, Belgium
UK Free Phone: 0800 169 31 47
France Free Phone:
0800 90 99 79
Germany Free Phone: 0800 181 99 10
Telephone:
+32 (0)2 653 0001
Fax:
+32 (0)2 653 0050
Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Telephone:
+81 3 5225 3638
Fax:
+81 3 5261 8733
Copyright 2009 Active Motif, Inc.
www.activemotif.com
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, in
whole 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
NFkB Transcription Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Transcription Factor Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
TransAM NFkB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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
NFkB Transcription Factor Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Appendix
Section A. Preparation of Nuclear Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section B. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Section C. Related Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
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Overview
The transcription factor NFκB (nuclear factor κB) is a key component for the inducible expression
of a wide variety of cellular and viral genes. Therefore, accurate monitoring of NFκB activation in
cells, 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 detect
and quantify transcription factor activation. TransAM Kits combine a fast, user-friendly format
with a sensitive, specific assay. TransAM NFκB p65, p50 and p52 Kits are designed specifically for
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 this oligonucleotide. By
using an antibody that is directed against either the NFκB p65, p50 or p52 subunit, the activated
NFκB subunit bound to the oligonucleotide is detected. Addition of a secondary antibody
conjugated to horseradish peroxidase (HRP) provides sensitive colorimetric readout that is easily
quantified by spectrophotometry. The 96-well plate with detachable wells is suitable for manual
use or high-throughput screening applications. TransAM NFκB p65, p52 and NFκB p50 Kits are
available in two sizes:
product
format
catalog no.
TransAM™ NFkB p65
1 x 96 rxns
5 x 96 rxns
40096
40596
TransAM™ NFkB p50
1 x 96 rxns
5 x 96 rxns
41096
41596
TransAM™ NFkB p52
1 x 96 rxns
5 x 96 rxns
48196
48596
The procedure outlined in this manual can be used for TransAM NFκB p65, TransAM NFκB p50 or
TransAM NFκB p52 Kits.
Active Motif also offers chemiluminescent versions of the TransAM NFκB p65, p50 and p52 Kits
that provide increased sensitivity and dynamic range, as well as the TransAM NFκB/Rel Family Kit
for the study of NFκB p50, p52, p65, c-Rel and RelB. See these and other Active Motif products
related to the NFκB signaling pathway in Appendix, Section C.
* 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
2
1
5
4
3
7
6
8
9
10
11
12
A
B
C
D
E
F
Cell extract
containing activated
transcription factor
G
H
Oligonucleotide
coated plate
Add cell
extract
21
11
01
9
8
7
6
5
4
3
2
1
A
B
C
D
E
F
G
H
Add primary
antibody
1 hr.
2
1
9
8
7
6
5
4
3
11
10
12
A
B
C
D
E
F
G
H
Add anti-IgG
HRP conjugate
21
11
01
9
8
7
6
5
1 hr.
4
3
2
1
A
B
C
D
E
F
G
Add developing
and stop solution
H
1 hr.
3
2
1
5
4
7
6
A
B
C
D
E
F
G
H
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8
9
10
11
12
Introduction
NFkB 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 a
variety of cellular and viral response genes2. NFκB is composed of homo- and heterodimeric
complexes of members of the Rel (NFκB) family. There are five subunits of the NFκB family in
mammals: p50, p65 (RelA), c-Rel, p52 and RelB3. These proteins share a conserved 300 amino
acid 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 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 found in the 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 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 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 C 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 incubated with 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, 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 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, typically
luciferase or β-galactosidase, placed under the control of a promoter containing the NFκB
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3
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 of this
procedure are: (i) reporter gene assays have to be repeated several times to obtain statistically reliable data; and (ii) reporter gene assays are sensitive to confounding factors that may
influence the expression level of the reporter gene. Therefore, assays have to be carefully
standardized. This method is sensitive and easy to perform with a large number of samples
but requires efficient cell transfection with the reporter plasmid.
TransAM NFkB
NFκB is a key factor in various immune processes and represents an excellent pharmacological target. 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 form
of 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 colorimetric readout that is easily quantified by spectrophotometry.
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 10-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 0.5 µg 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
Detection limit: < 0.5 µg nuclear extract/well. For whole-cell extract, twice as much input may be
needed. Alternatively, < 0.4 ng recombinant p50 or p65 protein/well may be used. TransAM NFκB
is 10-fold more sensitive than EMSA. (For increased sensitivity, the TransAM NFκB p50 Chemi,
TransAM NFκB p52 Chemi and TransAM NFκB p65 Chemi Kits utilize a chemiluminescent detection method that can detect 5 ng extract/well. See Appendix, Section C, Related Products.)
Range of detection: TransAM provides quantitative results from 0.2 to 10 µg of nuclear or wholecell extract per well or 0.1 to 40 ng of recombinant p50 or p65 protein/well.
Cross-reactivity:
p65
p50
p52
Human
+
+
+
Mouse
+
+
+
Rat
+
–
–
Assay time: 3.5 hours.
NFκB p65 activation (OD 450nm)
1.6
1.4
Jurkat
Jurkat+TPA+Cl
1.2
1
0.8
0.6
0.4
0.2
0
0.625
1.25
2.5
5
10
Extract (µg/well)
Monitoring NFkB activation with the TransAM NFkB Kit:
Nuclear extracts from Jurkat cells (Catalog No. 36014) and Jurkat cells stimulated with TPA and calcium ionophore (Catalog
No. 36013) were assayed from 0.625-10 µg/well for NFkB p65 activation using the TransAM NFkB p65 Kit. Data shown are
the results from wells assayed in duplicate. These results are provided for demonstration only.
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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 first
use. Then, we recommend storing each component at the temperature indicated in the table
below. All components are guaranteed stable for 6 months from date of receipt when stored
properly.
Reagents
Quantity
1 plate / 5 plates
Storage
NFkB p65 or p50 antibody
11 µl / 55 µl
-20°C for 6 months
NFkB 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
Developing Solution
11 ml / 55 ml
4°C for 6 months
Stop Solution
11 ml / 55 ml
4°C for 6 months
96-well NFkB 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 spectrophotometer capable of reading at 450 nm (655 nm as an optional
reference wavelength)
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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 (see the Quick
Chart for Preparing Buffers in this section). Some of the protease inhibitors lose their activity 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 form
clumps, 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.
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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 light
during storage. The Developing Solution may develop a yellow hue over time. This does not affect
product performance. A blue color present in the Developing Solution indicates that it has been
contaminated and must be discarded. Prior to use, place the Developing Solution at room temperature for at least 1 hour. Transfer the amount of Developing Solution required for the assay into
a secondary container before aliquoting into the wells (see the Quick Chart for Preparing Buffers
in this section). After use, discard remaining Developing Solution.
Stop Solution
Prior to use, transfer the amount of Stop Solution required for the assay into a secondary container (see the Quick Chart for Preparing Buffers in this section). After use, discard remaining Stop
Solution.
WARNING: The Stop Solution is corrosive. Wear personal protective equipment when
handling, i.e. safety glasses, gloves and labcoat.
Nuclear extract
The Jurkat (TPA + CI) nuclear extract is provided as a positive control for NFκB p50 and p65 activation.
This extract is optimized to give a strong signal when used at 2.5 µg/well. The Raji nuclear extract is
provided as a positive control for NFκB p52 activation and should be used at 5 µg/well. We recommend
aliquoting the extract in 5 µl fractions and storing at -80ºC. Avoid multiple freeze/thaw cycles of the
extract. Various cell extracts are available from Active Motif (see Appendix, Section C. Related Products).
Wild-type and mutated consensus oligonucleotides
The wild-type consensus oligonucleotide is provided as a competitor for NFκB binding in order to
monitor 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 the
required 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
the oligonucleotide 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.
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 C. Related Products).
Each protein is supplied with a lot specific Certificate of Analysis. Please refer to this before preparing the standard curve as the concentration of the protein varies from lot to lot.
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8
1. Begin with a 100 ng/µl working stock of recombinant protein (use the TransAM Complete Lysis 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. Refer to the protein’s lot specific data sheet for details.
2. Make up a 0.5 ng/µl solution by adding 1 µl of the 100 ng/µl working stock to 199 µl of Complete
Lysis Buffer. Next, pipette 60 µl of Complete Lysis Buffer into the 7 remaining tubes. Use the 0.5 ng/
µl solution to prepare a dilution series as indicated below. Mix tubes thoroughly before each transfer.
The 0.5 ng/µl standard is the high standard and Lysis Buffer alone is the 0.0 ng/µl.
60 µl
0.5 ng/µl
60 µl
0.25 ng/µl
60 µl
0.125 ng/µl
60 µl
0.0625 ng/µl
60 µl
0.0312 ng/µl
60 µl
0.0156 ng/µl
0.008 ng/µl
3. 20 µl from each tube will be aliquoted to the wells in Step 1, No. 2 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.
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 µl
0.23 µl
22.2 µl
22.5 µl
0.9 µl
1.8 µl
177.3 µl
180 µl
5.4 µl
10.8 µl
1.064 ml
1.08 ml
10.8 µl
21.6 µl
2.128 ml
2.16 ml
Complete Binding Buffer
DTT
Herring sperm DNA
Binding Buffer AM3
TOTAL REQUIRED
0.07 µl
0.34 µl
33.4 µl
33.8 µl
0.54 µl
2.7 µl
267 µl
270 µl
3.2 µl
16.2 µl
1.6 ml
1.62 ml
6.5 µl
32.4 µl
3.2 ml
3.24 ml
Complete Binding Buffer
with NFκB wild-type or
mutated oligonucleotide
NFκB wild-type or mutated oligo 2 µl
Complete Binding Buffer
31.8 µl
TOTAL REQUIRED
33.8 µl
16 µl
254 µl
270 µl
96 µl
1.524 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 µl
2.25 ml
16.2 ml
1.8 ml
18 ml
97.2 ml
10.8 ml
108 ml
194.4 ml
21.6 ml
216 ml
202.5 µl
22.5 µl
225 µl
1.62 ml
180 µl
1.8 ml
9.72 ml
1.08 ml
10.8 ml
19.44 ml
2.16 ml
21.6 ml
Reagents to prepare
Components
Complete Lysis Buffer
1X Antibody Binding Buffer* Distilled water
10X Ab Binding Buffer AM2
TOTAL REQUIRED
Developing Solution
TOTAL REQUIRED
112.5 µl
900 µl
5.4 ml
10.8 ml
Stop Solution
TOTAL REQUIRED
112.5 µl
900 µl
5.4 ml
10.8 ml
* Volumes listed refer to the preparation of buffer for diluting both the primary & secondary
antibodies.
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9
NFkB Transcription Factor Assay
Determine the appropriate number of microwell strips required for testing samples, controls and
blanks in duplicate. If less than 8 wells in a strip need to be used, cover the unused wells with a
portion of the plate sealer while you perform the assay. The content of these wells is stable at
room temperature if kept dry and, therefore, can be used later for a separate assay. Store the
unused strips in the aluminum pouch at 4°C. Use the strip holder for the assay.
Prepare the Complete Lysis Buffer, Complete Binding Buffer, 1X Wash Buffer and 1X Antibody
Binding Buffer as described above in the section Buffer Preparation and Recommendations. Multichannel pipettor reservoirs may be used for dispensing the Complete Binding Buffer, Wash Buffer,
Antibody Binding Buffer, Developing Solution and Stop Solution into the wells being used.
Step 1: Binding of NFkB to its Consensus Sequence
1. Add 30 µl Complete Binding Buffer to each well to be used. If you wish to perform competitive binding experiments, add 30 µl Complete Binding Buffer that contains 20 pmol (2 µl) of
the wild-type or mutated oligonucleotide (see the Buffer Preparation section above for a
description of competitive binding).
2.
Sample wells: Add 20 µl of sample diluted in Complete Lysis Buffer per well. We recommend using 2-20 µg of nuclear extract diluted in Complete Lysis Buffer per well. A protocol
for preparing nuclear extracts is provided on page 13. For whole-cell extracts we recommend
using 4-40 µg per well.
Positive control wells: For p65 or p50 add 2.5 µg of the provided Jurkat nuclear extract
(1 µl of nuclear extract in 19 µl of Complete Lysis Buffer per well). For p52 use 5 µg of the provided Raji nuclear extract diluted in 20 µl of Complete Lysis Buffer per well (2 µl of nuclear
extract in 18 µl of Complete Lysis Buffer per well).
Blank wells: Add 20 µl Complete Lysis Buffer only per well.
OPTIONAL – Protein standard wells for p50 and p65: Add 20 µl of the appropriate protein standard diluted in Complete Lysis Buffer to each well being used (see page 8, for details)
(Optional)
Blank Positive Control
Sample
(Optional) (Optional) Recombinant
Reagents
wells no competition no competition wild-type mutated
protein
Complete Binding Buffer
Wild-type oligonucleotide
Mutated oligonucleotide
Complete Lysis Buffer
Sample in Complete Lysis Buffer
Recombinant protein sample
3.
30 µl
–
–
20 µl
–
–
30 µl
–
–
–
20 µl
–
30 µl
–
–
–
20 µl
–
28 µl
2 µl
–
–
20 µl
–
28 µl
–
2 µl
–
20 µl
–
30 µl
–
–
–
–
20 µl
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.
www.activemotif.com
10
4.
Wash each well 3 times with 200 µl 1X Wash Buffer. For each wash, flick the plate over a sink
to empty the wells, then tap the inverted plate 3 times on absorbent paper towels.
Step 2: Binding of Primary Antibody
1.
Add 100 µl of diluted NFkB antibody (1:1,000 dilution in 1X Antibody Binding Buffer) to each
well 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 100 µl of diluted HRP-conjugated antibody (1:1,000 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).
Step 4: Colorimetric Reaction
1.
Add 100 µl Developing Solution to all wells being used.
2.
Incubate 30 seconds to 5 minutes at room temperature protected from direct light. Please
read the Certificate of Analysis supplied with this kit for optimal development time for
this specific kit lot. Monitor the blue color development in the sample wells until it turns
medium to dark blue. Do not overdevelop. NOTE: Recombinant standards develop quickly.
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 an optional
reference 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 optical density (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 and regression analysis may
also be applied.
To quantify the amount of NFκB in the samples, find the absorbance 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.
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11
Example curve:
The following standard curve is provided for demonstration only. A standard curve
should be made every time an experiment is performed.
NFκB p50 TransAM Assay
y = 0.468x - 0.055 r = 0.999
1.5
A450
1
A450
0.5
0
0
0.5
1
1.5
2
2.5
3
p50 Recombinant Protein (ng)
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.
www.activemotif.com
12
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 to
remove 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 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.
Check the cells under a microscope to monitor the cell lysis. The cell membrane should be
completely 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 blank
sample 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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13
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
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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14
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 the
correct order
Substrate or conjugate
is no longer active
Test conjugate and substrate for activity by mixing HRP and
Developing Solution together
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
Incorrect assay
temperature
Bring Developing Solution and Stop Solution to room temperature before using
Inadequate volume of
Developing Solution
Check to make sure that correct volume is delivered by pipette
Measurement time
too long
Stop enzymatic reactions as soon as the positive wells turn
medium-dark blue
Concentration of antibodies is 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 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 1:2,000 for primary antibody and
1:5,000 for the secondary antibody. The sensitivity of the assay
will be decreased
Not enough nuclear
extract per well
Increase amount of nuclear extract. Not to exceed 50 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
NFkB is poorly activated or inactivated in
nuclear fractions
Perform a time course for NFkB 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.
Salt concentration
too high in binding
reaction
Reduce amount of extract per well or dialyze extract before
use
High background in
all wells
Uneven color
development
High background in
sample wells
No signal or weak signal
in sample wells
www.activemotif.com
15
Section C. Related Products
Transcription Factor ELISAs
Format
Catalog No.
TransAM™ AML-1/Runx1
1 x 96-well plate
47396
1 x 96-well plate
44496
TransAM™ AML-3/Runx2
2 x 96-well plates
44296
TransAM™ AP-1 Family
1 x 96-well plate
44096
TransAM™ AP-1 c-Fos
1 x 96-well plate
46096
TransAM™ AP-1 c-Jun
1 x 96-well plate
45096
TransAM™ AP-1 FosB
1 x 96-well plate
43496
TransAM™ AP-1 JunD
1 x 96-well plate
42396
TransAM™ ATF-2
1 x 96-well plate
43396
TransAM™ c-Myc
1 x 96-well plate
44196
TransAM™ C/EBP a/b
1 x 96-well plate
42096
TransAM™ CREB
1 x 96-well plate
43096
TransAM™ pCREB
1 x 96-well plate
44396
TransAM™ Elk-1
1 x 96-well plate
41396
TransAM™ ER
1 x 96-well plate
46396
TransAM™ FKHR (FOXO1/4)
2 x 96-well plates
48296
TransAM™ GATA Family
1 x 96-well plate
46496
TransAM™ GATA-4
1 x 96-well plate
45496
TransAM™ GR
1 x 96-well plate
47096
TransAM™ HIF-1
2 x 96-well plates
46296
TransAM™ HNF Family
1 x 96-well plate
46196
TransAM™ HNF-1
1 x 96-well plate
48396
TransAM™ IRF-3 (Human)
1 x 96-well plate
48496
TransAM™ IRF-3 (Mouse)
1 x 96-well plate
50196
TransAM™ IRF-7
2 x 96-well plates
47296
TransAM™ MAPK Family
1 x 96-well plate
43196
TransAM™ MEF2
1 x 96-well plate
47196
TransAM™ MyoD
1 x 96-well plate
40396
TransAM™ NF-YA
1 x 96-well plate
40296
TransAM™ NFATc1
2 x 96-well plates
43296
TransAM™ NFkB Family
2 x 96-well plates
43298
TransAM™ Flexi NFkB Family
1 x 96-well plate
41096
TransAM™ NFkB p50
1 x 96-well plate
41097
TransAM™ NFkB p50 Chemi
1 x 96-well plate
41098
TransAM™ Flexi NFkB p50
1 x 96-well plate
48196
TransAM™ NFkB p52
1 x 96-well plate
48197
TransAM™ NFkB p52 Chemi
1 x 96-well plate
40096
TransAM™ NFkB p65
1 x 96-well plate
40097
TransAM™ NFkB p65 Chemi
1 x 96-well plate
40098
TransAM™ Flexi NFkB p65
1 x 96-well plate
50296
TransAM™ Nrf2
1 x 96-well plate
42496
TransAM™ Oct-4
1 x 96-well plate
41196
TransAM™ p53
1 x 96-well plate
40196
TransAM™ PPARg
1 x 96-well plate
41296
TransAM™ Sp1
1 x 96-well plate
40496
TransAM™ Sp1/Sp3
2 x 96-well plates
42296
TransAM™ STAT Family
1 x 96-well plate
45196
TransAM™ STAT3
1 x 96-well plate
51396
TransAM™ T-bet
For a complete, up-to-date list of available TransAM™ Kits, please visit www.activemotif.com/transam
Function ELISA
FunctionELISA IkBa
www.activemotif.com
16
Format
Catalog No.
1 x 96-well plate
48005
Recombinant Proteins
Format
Catalog No.
Recombinant c-Fos protein
Recombinant c-Jun protein
Recombinant c-Myc protein
Recombinant CREB protein
Recombinant elF2a protein
Recombinant NFkB p50 protein
Recombinant NFkB p65 protein
Recombinant p53 protein
Recombinant p300 protein
Purified Sp1 protein
Recombinant STAT3 protein
5 µg
5 µg
5 µg
25 µg
25 µg
5 µg
5 µg
5 µg
4 µg
2 µg
10 µg
31115
31116
31117
31107
31108
31101
31102
31103
31124
31137
31140
Histone ELISAs
Format
Catalog No.
Histone H3 monomethyl Lys4 ELISA
Histone H3 dimethyl Lys4 ELISA
Histone H3 trimethyl Lys4 ELISA
Histone H3 dimethyl Lys9 ELISA
Histone H3 trimethyl Lys9 ELISA
Histone H3 monomethyl Lys27 ELISA
Histone H3 trimethyl Lys27 ELISA
Histone H3 phospho Ser10 ELISA
Histone H3 phospho Ser28 ELISA
Total Histone H3 ELISA
1 x 96 rxns
1 x 96 rxns
1 x 96 rxns
1 x 96 rxns
1 x 96 rxns
1 x 96 rxns
1 x 96 rxns
1 x 96 rxns
1 x 96 rxns
1 x 96 rxns
53101
53112
53113
53108
53109
53104
53106
53111
53100
53110
Histone Purification & Chromatin Assembly
Format
Catalog No.
Histone Purification Kit
Histone Purification Mini Kit
Chromatin Assembly Kit
HeLa Core Histones
10 rxns
10 rxns
10 rxns
36 µg
40025
40026
53500
53501
Co-Immunoprecipitation
Format
Catalog No.
Nuclear Complex Co-IP Kit
Universal Magnetic Co-IP Kit
50 rxns
25 rxns
54001
54002
DNA Methylation
Format
Catalog No.
MethylDetector™
MethylCollector™
MethylCollector™ Ultra
UnMethylCollector™
Fully Methylated Jurkat DNA
50 rxns
25 rxns
30 rxns
30 rxns
10 µg
55001
55002
55005
55004
55003
SUMOylation
Format
Catalog No.
SUMOlink™ SUMO-1 Kit
SUMOlink™ SUMO-2/3 Kit
20 rxns
20 rxns
40120
40220
www.activemotif.com
17
ChIP-validated Antibodies
Application
AP-2 pAb
ChIP, EMSA
ChIP, EMSA, IF
c-Jun pAb
ChIP, EMSA, IF, WB
C/EBPa pAb
ChIP, WB
CTCF mAb
ChIP, IHC, IP, WB
DNMT1 mAb
ChIP, IF, IHC, WB
DNMT3A mAb
ChIP, IF, IP, WB
DNMT3B mAb
ChIP, EMSA
E2F-1 pAb
ChIP, WB
E2F-6 mAb
ChIP, WB
EZH2 pAb
ChIP, IF, WB
HBP-1 mAb
ChIP, IF, IHC, IP, WB
HDAC1 mAb (Clone 10E2)
ChIP, IF, IHC, IP, WB
HDAC2 mAb (Clone 3F3)
ChIP, WB
HDAC3 pAb
ChIP, WB
HDAC4 pAb
ChIP, WB
HDAC5 pAb
ChIP, WB
HDAC6 pAb
ChIP, WB
Histone H2A pAb
ChIP, IF, IP, WB
Histone H2A phospho Ser129 pAb
ChIP, WB
Histone H2A.Z pAb
ChIP, WB
Histone H2B pAb
ChIP, WB
Histone H2B acetyl Lys5 pAb
ChIP, WB
Histone H2B acetyl Lys16 pAb
ChIP, WB
Histone H2B acetyl Lys46 pAb
ChIP, WB
Histone H3, C-terminal pAb
ChIP, IF, WB
Histone H3 acetyl Lys4 pAb
ChIP, WB
Histone H3 monomethyl Lys4 mAb
ChIP, WB
Histone H3 dimethyl Lys4 pAb
ChIP, WB
Histone H3 trimethyl Lys4 pAb
ChIP, IF, WB
Histone H3 dimethyl Lys9 pAb
ChIP, WB
Histone H3 trimethyl Lys9 pAb
ChIP, IF, WB
Histone H3 acetyl Lys18 pAb
ChIP, IF, WB
Histone H3 acetyl Lys27 pAb
ChIP, WB
Histone H3 acetyl Lys27 pAb
ChIP, IF, WB
Histone H3 dimethyl Lys27 pAb
ChIP, WB
Histone H3 trimethyl Lys27 mAb
ChIP, IF, WB
Histone H3 trimethyl Lys27 pAb
ChIP, WB
Histone H3 trimethyl Lys27 pAb
ChIP, IF, WB
Histone H3 acetyl Lys36 pAb
ChIP, WB
Histone H3 acetyl Lys56 pAb
ChIP, IF, WB
Histone H3 acetyl Lys64 pAb
ChIP, WB
Histone H3 acetyl Lys79 pAb
ChIP, IF, WB
Histone H4 pan-acetyl pAb
ChIP, WB
Histone H4 tetra-acetyl pAb
ChIP, IF, WB
Histone H4 acetyl Lys5 pAb
ChIP, IF, WB
Histone H4 acetyl Lys5 pAb
ChIP, IF, WB
Histone H4 acetyl Lys12 pAb
ChIP, WB
Histone H4 acetyl Lys16 pAb
ChIP, IF, WB
Histone H4 monomethyl Lys20 pAb
ChIP, IF, WB
Histone H4 trimethyl Lys20 pAb
ChIP, WB
IRF-3 pAb
ChIP, EMSA
JunB pAb
ChIP, EMSA
JunD pAb
ChIP, IP, WB
L3MBTL1 pAb
ChIP, EMSA
p53 pAb
www.activemotif.com
18
Format
Catalog No.
17 rxns
100 µg
100 µg
200 µg
100 µg
100 µg
100 µg
17 rxns
100 µl
100 µg
100 µl
200 µl
200 µl
100 µg
100 µg
100 µg
100 µg
200 µl
200 µl
200 µl
200 µl
200 µl
200 µl
200 µl
200 µl
200 µl
100 µg
200 µl
200 µl
200 µl
200 µl
200 µl
200 µg
200 µl
200 µl
200 µl
200 µg
200 µl
200 µl
200 µl
200 µl
200 µl
200 µl
50 µl
200 µl
200 µl
200 µl
200 µl
200 µl
200 µl
100 µl
17 rxns
100 µl
200 µl
17 rxns
39304
39309
39306
39621
39204
39206
39207
39313
39509
39639
39511
39531
39533
40968
40969
40970
40971
39235
39271
39113
39237
39123
39121
39571
39163
39381
39635
39141
39159
39239
39161
39587
39133
39135
39245
39535
39155
39156
39379
39281
39545
39565
39243
39179
39169
39583
39165
39167
39175
39180
39033
39326
39328
39182
39334
PP2A pAb
ChIP, IP, WB
200 µl
39192
ChIP, WB
200 µl
39198
RbAp46/48 pAb
ChIP, ELISA, IF, IP, WB
200 µl
39097
RNA pol II mAb
ChIP, IF, IP, WB
200 µl
39543
SNF2h mAb
ChIP, WB
100 µl
39058
Sp1 pAb
ChIP, IP, WB
100 µg
39223
TRF2 Goat pAb
For an up-to-date list of ChIP-validated antibodies, please visit www.activemotif.com/chipabs
Application Key: ChIP = Chromatin Immunoprecipitation; EMSA = Electrophoretic Mobility Shift Assay;
IF = Immunofluorescence; IHC = Immunohistochemistry; IP = Immunoprecipitation; WB = Western blot
ChIP-IT™ Kits
Format
Catalog No.
ChIP-IT™ Express
ChIP-IT™ Express Enzymatic
ChIP-IT™ Express HT
Re-ChIP-IT™
ChIP-IT™
ChIP-IT™ Enzymatic
ChIP-IT™ Shearing Kit
Enzymatic Shearing Kit
ChIP-IT™ Protein G Magnetic Beads
Salmon Sperm DNA/Protein G agarose
ChIP-IT™ Control Kit – Human
ChIP-IT™ Control Kit – Mouse
ChIP-IT™ Control Kit – Rat
Ready-to-ChIP HeLa Chromatin
Ready-to-ChIP Hep G2 Chromatin
Ready-to-ChIP K-562 Chromatin
Ready-to-ChIP NIH/3T3 Chromatin
25 rxns
25 rxns
96 rxns
25 rxns
25 rxns
25 rxns
10 rxns
10 rxns
25 rxns
25 rxns
5 rxns
5 rxns
5 rxns
10 rxns
10 rxns
10 rxns
10 rxns
53008
53009
53018
53016
53001
53006
53002
53005
53014
53003
53010
53011
53012
53015
53019
53020
53021
www.activemotif.com
19
Technical Services
If you need assistance at any time, please call Active Motif Technical Service at one of the
numbers listed below.
Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008
USA
Toll Free:
877 222 9543
Telephone: 760 431 1263
Fax:
760 431 1351
E-mail:
[email protected]
Active Motif Europe
104 Avenue Franklin Roosevelt
B-1330 Rixensart, Belgium
UK Free Phone:
0800 169 31 47
France Free Phone: 0800 90 99 79
Germany Free Phone: 0800 181 99 10
Telephone: +32 (0)2 653 0001
Fax: +32 (0)2 653 0050
E-mail: [email protected]
Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Telephone: +81 3 5225 3638
Fax:
+81 3 5261 8733
E-mail:
[email protected]
Visit Active Motif on the worldwide web at http://www.activemotif.com
At this site:
• Read about who we are, where we are, and what we do
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20