TransAM™ c-Myc

Transcription Factor Assay Kits
(version A5)
Catalog Nos. 43396 & 43896

Copyright 2009 Active Motif, Inc.

Revision

Date

Description of Change

A5

July 17, 2023

c-Myc antibody has changed to new monoclonal
antibody. However, dilution used in protocol andspecies reactivity are the same as previous.

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
c-Myc Transcription Factor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Transcription Factor Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
TransAM c-Myc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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
c-Myc Transcription Factor Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Preparation of Nuclear Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Appendix
Section A. Troubleshooting Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

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Overviewc-Myc is a transcription factor that regulates cell growth and differentiation, glycolysis and apoptosis.1 Deregulation of c-Myc has been implicated in the origin of diverse human cancers, andmay contribute to as much as one-seventh of U.S. cancer deaths.2 Because of this link between
c-Myc and cancer, accurate monitoring of c-Myc in cells, tissues and animals is crucial for many
biomedical research and drug development projects. To date, such research projects are tediousand time consuming, and lack high-throughput screening methods.
With its patented TransAM™ method*, Active Motif introduced the first ELISA-based kits to detectand quantify transcription factor activation. TransAM Kits combine a fast, user-friendly format witha sensitive, specific assay. TransAM c-Myc Kits are designed specifically to detect and quantify
c-Myc activation. They contain a 96-well plate to which oligonucleotide containing a c-Myc
consensus sequence has been immobilized. c-Myc dimers contained in nuclear extracts bind
specifically to this oligonucleotide and are detected through use of an antibody directed against
c-Myc. Addition of a secondary antibody conjugated to horseradish peroxidase (HRP) provides asensitive colorimetric readout that is easily quantified by spectrophotometry. The 96-well platewith individual strips of 8 wells is suitable for manual use or for high-throughput screening applications. TransAM c-Myc Kits are available in two sizes:
product
TransAM c-Myc

formatcatalog no.

1 x 96-well plate5 x 96 well plates

43396
43896

Active Motif also offers the TransAM MAPK Family Kit that directly assays the transcription factors
ATF-2, c-Jun, c-Myc, MEF2 and STAT1, all of which are regulated by MAP kinase cascades. See
this and other Active Motif products related to c-Myc in Appendix, Section B.

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

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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
F

Cell extractcontaining activatedtranscription 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.
3

2

1

9

8

7

6

5

4

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
c-Myc Transcription Factor
c-Myc was originally discovered as the cellular homolog of the retroviral v-myc oncogene2, and isa transcription factor involved in a wide variety of cellular processes, including cell proliferation,
replicative potential, growth, differentiation and apoptosis.1 Expression of c-Myc is induced by
mitogenic signals and suppressed by growth-inhibitory signals.3 c-Myc is a member of the basic
helix-loop-helix leucine zipper (bHLHzip) family, along with B-Myc, N-Myc, L-Myc and s-Myc.2
Upon dimerization with the bHLHzip protein Max, c-Myc can bind to the E box motif CACGTG andactivate transcription. The gene for c-Myc is located on human chromosome 8 and is comprisedof three exons.2 c-Myc is a 64 kDa protein4 that has an N-terminal transactivation/repression
domain and a C-terminal bHLH-LZ DNA binding domain.3 c-Myc is phosphorylated at Ser62, which
has been shown to be a regulatory site of phosphorylation. The phosphorylation of c-Myc causes
increased function of the NH2-terminal transactivation domain, and studies have indicated thatthe expression of MAP kinase is responsible for increased c-Myc phosphorylation at Ser62.5
Because of the central role of c-Myc as an activator of diverse cellular processes, regulation ofthis transcription factor is crucial for proper cell function and ultimate survival. The main regulation of c-Myc occurs through its binding with the bHLHzip protein Max, which can also form
heterodimers with members of the Mad family (Mad 1, 3, 4 and Mxi1). As c-Myc cannot bind to
DNA without forming a heterodimer with Max, competition between c-Myc and Mad for the common partner Max is used to regulate c-Myc activity. While Max is a relatively stable protein, c-Myc
degrades rapidly, with a half-life of 20-30 minutes.3
c-Myc regulates a wide variety of genes involved in the cell cycle, glycolysis and apoptosis,
including cell cycle regulators CDC2-L1 and cyclin B1, and glycolysis regulator lactate dehydrogenase A.1 c-Myc also mediates induction of transcription cofactors and components of DNA
pol II, facilitating an overall increase in transcriptional activity during the G1/S transition. Genes
associated with terminal differentiation such as NCAM, gadd45, neu and adrenomedullin can be
repressed by c-Myc. Activation of c-Myc by proviral insertion, gene amplification and chromosomal translocation has been described in a variety of tumors.3 The c-myc gene is amplified invarious human cancers, with an elevated expression found in almost one-third of breast andcolon cancers.2 It has been estimated that approximately 70,000 U.S. cancer deaths per year areassociated with changes in the c-myc gene or its expression.2
Transcription Factor Assays
To date, three methods are widely used to measure c-Myc activation, either directly or indirectly:1.

c-Myc activation can be determined by Western blot by using antibodies specific for c-Myc
proteins. This method is time consuming (up to 2 days once the nuclear extracts are prepared), and is not suitable for processing large numbers of samples.

2.

The DNA-binding capacity of c-Myc can be assayed by gel retardation, also called electrophoretic mobility shift assay (EMSA). In this method, nuclear extracts are incubated with a radioactive
double-stranded oligonucleotide probe containing the consensus sequence for c-Myc binding.
If c-Myc is active in the nuclear extract, it will bind to the probe. Samples are then resolved byelectrophoresis on a native polyacrylamide gel, followed by autoradiography. This method iswww.activemotif.com

3

sensitive, but like the previous procedure, it is time consuming (multiple days of gel exposure
may be required to achieve sufficient sensitivity) and cannot be applied to high-throughput
screening. Gelshift assays also require special precautions and equipment for handling radioactivity.
3.

Another method used to assay c-Myc activation is based on reporter genes, typically luciferase
or β-galactosidase, placed under the control of a promoter containing the c-Myc recognition site.
The promoter can be artificial, made of several c-Myc elements or natural, like the LDHA promoter
sequence. However, the procedure is limited by the following issues: (i) reporter gene assays haveto 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, andtherefore assays have to be carefully standardized. Reporter gene assays are sensitive and easy
to perform with a large number of samples, but require efficient cell transfection with the reporter
plasmid.

TransAM c-Myc
c-Myc is a central transcription factor that regulates cell growth and differentiation, glycolysis
and apoptosis. Aberrant c-Myc signaling can affect the outcome of these pathways and result in
oncogenesis, which makes it an excellent pharmacological target. However, this field has beenhampered by the lack of convenient assays suitable for detecting c-Myc and for performing highsample number experiments.
To help achieve this, Active Motif offers a high-throughput assay to quantify c-Myc activation. The
TransAM c-Myc Kit combines a fast and user-friendly ELISA format with a sensitive and specificassay for transcription factors. TransAM c-Myc Kits contain a 96-well plate on which has been
immobilized an oligonucleotide that contains the c-Myc consensus binding site (5´-CACGTG-3´).
The active form of c-Myc contained in nuclear extract binds specifically to this oligonucleotide.
The primary antibody used in the TransAM c-Myc Kit recognizes an accessible epitope on c-Myc
protein upon DNA binding. Addition of a secondary HRP-conjugated antibody provides a sensitive
colorimetric readout easily quantified by spectrophotometry. Once the nuclear extracts are prepared, this assay is completed in less than 3.5 hours. As this assay is performed in 96-well plates,
a large number of samples can be handled simultaneously, enabling high-throughput automation.
This assay is specific for c-Myc activation and has been shown to be 20-fold more sensitive and20-fold faster than the gel-retardation technique. With the 3.5-hour TransAM procedure, we could
detect c-Myc activation using as little as 0.25 µg of nuclear extract from Jurkat cells. A comparable assay using EMSA required 5 µg of nuclear extract and a 3-day autoradiography.
TransAM has many applications including the study of drug potency, inhibitor or activator proteins, and protein structure/function studies in the c-Myc signaling pathway.

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4Kit Performance and Benefits
TransAM c-Myc Kits are for research use only. Not for use in diagnostic procedures.
Detection limit: < 0.25 µg nuclear extract/well. TransAM c-Myc is 20-fold more sensitive than
EMSA.
Range of detection: TransAM provides quantitative results from 0.25 to 5 µg of nuclear extract/well (see graph below).
Cross-reactivity: The TransAM c-Myc Kit detects c-Myc from human and mouse origin.

c-Myc activation (OD 450nm)

Assay time: 3.5 hours. TransAM is 20-fold faster than EMSA.

0.8

U-937
Jurkat

0.6
0.4
0.2
0

0.3125

0.625

1.25

Extract (µg/well)

2.5

5

Monitoring c-Myc activity with the TransAM c-Myc Kit: Different amounts of nuclear extracts
from untreated Jurkat (1 day growth) and U-937 cells are tested for c-Myc activity by using theTransAM c-Myc Kit. This data is provided for demonstration only.

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5Kit Components and Storage
Except for the nuclear extract that must be kept at -80ºC, kit components can be stored at -20ºCprior to first use. Then, we recommend storing each component at the temperature indicated in
the table below.
Quantity
1 plate / 5 plates

Storage / Stability

c-Myc antibody

11 µl / 55 µl-20°C for 6 months

Anti-rabbit HRP-conjugated antibody

11 µl / 55 µl(0.2 µg/µl)

4°C for 6 months

Wild-type oligonucleotide AM3

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

Mutated oligonucleotide AM3

100 µl / 500 µl(10 pmol/µl)

Jurkat (1 day growth) nuclear extract

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

Dithiothreitol (DTT)

100 µl / 500 µl(1 M)-20ºC for 6 months

Protease Inhibitor Cocktail

100 µl / 500 µl-20ºC for 6 months

Lysis Buffer AM1

10 ml / 50 ml

4°C for 6 months

Binding Buffer AM6

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 AM3

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 c-Myc assay plate

1/5

4°C for 6 months

Plate sealer

1/5

Reagents´-20ºC for 6 months

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 optionalreference wavelength)www.activemotif.com

6

Protocols
Buffer Preparation and Recommendations
Preparation of Complete Lysis Buffer
We provide an excess of Lysis Buffer AM1 in order to perform the assay AND to prepare customized nuclear extracts. Our Nuclear Extract Kit can also be purchased separately (Cat. Nos. 40010& 40410). Prepare the amount of Complete Lysis Buffer required for the assay by adding 1 µl of
1 M DTT and 10 µl Protease Inhibitor Cocktail per ml of Lysis Buffer AM1 (see the Quick Chart for
Preparing Buffers in this section). Some of the protease inhibitors lose their activity after 24 hours
once diluted. Therefore, we recommend using the Complete Lysis Buffer immediately for cell
lysis. The remaining amount should be discarded if not used in the same day.
Preparation of Complete Binding Buffer
Prepare the amount of Complete Binding Buffer required for the assay by adding 2 µl of 1 M DTT
per ml of Binding Buffer AM6 (see the Quick Chart for Preparing Buffers in this section). After use,
discard the 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, which makes it necessary to homogenize the buffer by incubating at 50ºC for 2 minutes
and mixing prior to use.
Preparation of 1X Antibody Binding Buffer
Prepare the amount of 1X Antibody Binding Buffer required for the assay as follows: For every
10 ml of 1X Antibody Binding Buffer required, dilute 1 ml 10X Antibody Binding Buffer AM3 with
9 ml distilled water (see the Quick Chart for Preparing Buffers in this section)*. Mix gently to
avoid foaming. Discard remaining 1X Antibody Binding Buffer after use. The BSA contained in
10X Antibody Binding Buffer AM3 may form clumps, therefore you should homogenize the buffer
by warming to room temperature and vortexing for 1 minute prior to use. Dilute the primary andsecondary antibodies with the 1X Antibody Binding Buffer to 1:1000. Depending on the particular
assay, the signal:noise ratio may be optimized by using higher dilutions of both antibodies. This
may decrease the sensitivity of the assay.
* Volumes listed refer to the preparation of buffer for diluting both the primary & secondary antibodies.

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7Developing Solution
The Developing Solution should be warmed to room temperature before use. The Developing
Solution is light sensitive, therefore, we recommend avoiding direct exposure to intense lightduring storage. The Developing Solution may develop a yellow hue over time. This does notaffect product performance. A blue color present in the Developing Solution indicates that it has
been contaminated and must be discarded. Prior to use, place the Developing Solution at room
temperature for at least 1 hour. Transfer the amount of Developing Solution required for the assay
into a secondary container before aliquoting into the wells (see the Quick Chart for Preparing
Buffers in this section). After use, discard remaining Developing Solution.
Stop Solution
Prior to use, transfer the amount of Stop Solution required for the assay into a secondary
container (see the Quick Chart for Preparing Buffers in this section). After use, discard remaining
Stop Solution.
WARNING: The Stop Solution is corrosive. Wear personal protective equipment whenhandling, (i.e. safety glasses, gloves and labcoat).
Jurkat nuclear extract
The Jurkat (1 day growth) nuclear extract is provided as a positive control for c-Myc activation.
Nuclear extract was made from unstimulated Jurkat cells which were seeded and grown for 24hours prior to harvesting. Sufficient extract is supplied for 20 reactions. This extract is optimized togive a strong signal when 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 areavailable from Active Motif (see Appendix, Section B. Related Products).
Wild-type and mutated consensus oligonucleotides
The wild-type consensus oligonucleotide is provided as a competitor for c-Myc binding in order tomonitor the specificity of the assay. Used at 20 pmol/well, the oligonucleotide will prevent c-Myc
binding to the probe immobilized on the plate. Conversely, the mutated consensus oligonucleotide should have no effect on c-Myc binding. Prepare the required amount of wild-type and/or
mutated consensus oligonucleotide by adding 2 µl of appropriate oligonucleotide to
43 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 prior toaddition of the nuclear extract.
Optional- Preparation of standard curve
For those who wish to quantify the amount of c-Myc in their samples, Active Motif offers recombinant c-Myc for use as a protein standard (see Appendix, Section B. Related Products).
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 and 0 ng/µl. Note: The preceding range is provided
as guidance, a broader range of values may be used.

2.

Make up a 0.5 ng/µl solution by adding 1.0 µl of the 100 ng/µl working stock to 199 µl ofComplete Lysis Buffer. Next, pipette 25 µl of Complete Lysis Buffer into the 3 remaining
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8

tubes. Use the 0.5 ng/µl solution to prepare a dilution series as indicated below. Be sure tomix each tube thoroughly before each transfer. The 0.5 ng/µl standard serves as the highstandard, while Complete Lysis Buffer alone serves as the 0.0 ng/µl.
60 l

0 5 ng l

3.

60 l

0 25 ng l

60 l

0 125 ng l

60 l

0 0625 ng l

60 l

0 0312 ng l

60 l

0 0156 ng l

0 008 ng l

10 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 c-Myc: 5, 2.5, 1.25, 0.625 and 0.0 ng/well.

Quick Chart for Preparing Buffers
Reagents to prepare

Components

For 1
well

For 1 strip(8 wells)

For 6 strips(48 wells)

For 12 strips(96 wells)

Complete Lysis Buffer

DTT
Protease Inhibitor Cocktail
Lysis Buffer AM1
TOTAL REQUIRED

0.01 µl0.12 µl11.12 µl11.25 µl

0.1 µl0.9 µl89.0 µl90.0 µl

0.6 µl5.4 µl534.0 µl
540.0 µl

1.2 µl10.8 µl
1.068 ml
1.08 ml

Complete Binding Buffer

DTT
Binding Buffer AM6
TOTAL REQUIRED

0.09 µl44.9 µl45 µl0.72 µl359.3 µl
360 µl4.32 µl2.15 ml
2.16 ml

8.64 µl
4.31 ml
4.32 ml

Complete Binding Buffer
with wild-type ormutated oligonucleotide

Wild-type or mutated oligo
Complete Binding Buffer
TOTAL REQUIRED

2 µl
43 µl
45 µl

16 µl
344 µl360 µl96 µl2.158 ml
2.16 ml

N/A
N/A
N/A

1X Wash Buffer

Distilled water
10X Wash Buffer AM2
TOTAL REQUIRED

2.025 ml
225.0 µl2.25 ml

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

194.4 ml21.6 ml
216.0 ml

1X Antibody
Binding Buffer*

Distilled water
10X Ab Binding Buffer AM3
TOTAL REQUIRED

202.5 µl22.5 µl225.0 µl

1.62 ml
180 µl
1.8 ml9.72 ml
1.08 ml
10.8 ml19.44 ml
2.16 ml21.6 ml

Developing Solution

TOTAL REQUIRED

112.5 µl900.0 µl

5.4 ml10.8 ml

Stop Solution

TOTAL REQUIRED

112.5 µl900.0 µl

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

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9c-Myc 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, 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 c-Myc to its consensus sequence1.

Add 40 µl Complete Binding Buffer to each well to be used. If you wish to perform competitive binding experiments, add 40 µl Complete Binding Buffer that contains 20 pmol (2 µl) of the wild-type or mutated consensus
oligonucleotide (see the Buffer Preparation section above for a description of competitive binding).

2.

Sample wells: Add 10 µl of sample diluted in Complete Lysis Buffer per well. A protocol for
preparing nuclear extracts can be found on page 12.
Positive control wells: Add 5 µg of the provided Jurkat nuclear extract diluted in 10 µl of Complete Lysis Buffer per well (2 µl of nuclear extract in 8 µl of Complete Lysis Buffer per well).
Blank wells: Add only 10 µl Complete Lysis Buffer per well.
OPTIONAL – Protein standard wells: Add 10 µl of the appropriate protein standard dilutedin Complete Lysis Buffer to each well being used (see page 8, Preparation of standard
curve).

3.

Use the provided adhesive cover to seal the plate. Incubate for 1 hour at room temperature
with mild agitation (100 rpm on a rocking platform).

4.

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

Step 2: Binding of primary antibody1.

Add 100 µl diluted c-Myc 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).

Step 3: Binding of secondary antibody1.

Add 100 µl of diluted HRP-conjugated antibody (1:1000 dilution in 1X Antibody Binding Buffer) to all wells being used.

2.

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

3.

During this incubation, place the Developing Solution at room temperature.

4.

Wash the wells 4 times with 200 µl 1X Wash Buffer (as described in Step 1, No. 4).
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10Step 4: Colorimetric reaction1.

Add 100 µl Developing Solution to all wells being used.

2.

Incubate 2-10 minutes at room temperature protected from direct light. Please read the Certificate of Analysis supplied with this kit for the optimal development time for this specific
kit lot, which varies from lot to lot. Monitor the blue color development in the sample andpositive control wells until it turns medium to dark blue. Do not overdevelop.

3.

Add 100 µl Stop Solution. In the presence of the acid, the blue color turns yellow.

4.

Read absorbance on a spectrophotometer within 5 minutes at 450 nm with a reference
wavelength of 655 nm. Blank the plate reader according to the manufacturer’s instructions
using the blank wells.

OPTIONAL – Calculation of results using the standard curve
If you have generated a standard curve using Active Motif’s recombinant c-Myc 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 c-Myc 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.
Example curve:
The following standard curve is provided for demonstration only. A standard curve should bemade every time an experiment is performed.
FκB p50 ransA Assayy 0 468x - 0 055 r 0 999

15

A450

1
A450

05

0
0

05

1

15

2

25

p50 Recombinant Protein ng

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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 TransAM Kits, which serves to reduce inconsistencies in
the assay that may arise from using homemade or other buffers. If you prefer to make your own
buffers, please refer to the following protocol.
This procedure can be used for a confluent cell layer of 75 cm2 (100-mm dish). The yield is approximately 0.15 mg of nuclear proteins for 9 x 106 cells.
1.

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

2.

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

3.

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

4.

Allow the cells to swell on ice for 15 minutes.

5.

Add 50 µl 10% Nonidet P-40 (0.5 % final) and vortex the tube vigorously for 10 seconds.

6.

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

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 –80ºC. 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 –20ºC.

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12PBS/PIB
Prior to use, add 0.5 ml PIB to 10 ml 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.

References
1.
2.
3.
4.
5.

Menssen A. et al. (2002) PNAS 99: 6274-6279.
Dang C.V. (1999) Molecular & Cellular Biology 19: 1-11.
Gregory M.A. et al. (2000) Molecular & Cellular Biology 20: 2423-2435.
Semmes O.J. et al. (1996) J. Biol. Chem. 271(16): 9730-9738.
Davis R.J. (1993) J. Biol. Chem. 268(20): 14553-14556.

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13

Appendix
Section A. 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
Developing Solution together

Enzyme inhibitor
present

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

Plate reader settings
not optimal

Verify the wavelength and filter settings

Incorrect assay
temperature

Bring Developing Solution and Stop Solution to room temperature before using

Inadequate volume ofDeveloping 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 highIncrease antibody dilutions

Inadequate washing

Ensure all wells are filled with Wash Buffer and follow washingrecommendations

Incomplete washingof wells

Ensure all wells are filled with Wash Buffer and follow washingrecommendations

Well cross-contamination

Follow washing recommendations

Too much nuclear
extract per well

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

Concentration of antibodies is too high

Perform antibody titration to determine optimal working
concentration. Start using 1:2,000 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

c-Myc is poorly activated or inactivated innuclear fractions

Perform a time course for c-Myc activation in the studied cell
line

Nuclear extracts
are not from correct
species

Perform study with a human or mouse model.

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