TransAM™

ER Transcription Factor
Assay Kits
(version C3)
Catalog Nos. 41396 & 41996

Copyright 2011 Active Motif, Inc.

Revision History
Revision

Date

Description of Change

C2

June 2019

Corrected recipe for Complete Lysis Buffer

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.
© 2011 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
ER Transcription Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Transcription Factor Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
TransAM ER. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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
ER Transcription Factor Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Appendix
Section A. Preparation of Nuclear Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section B. Troubleshooting Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

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Overview
The Estrogen Receptor (ER) is expressed in two forms, ERα and ERβ. These are encoded by two
distinct genes and are members of the steroid/thyroid hormone superfamily of nuclear receptors1.
The activity of these proteins (dimerization, DNA binding and interaction with other transcription
factors) is modulated by their corresponding ligands. Estrogens and estrogen-like molecules exert their effects through binding to and activating ER2. They affect growth, differentiation and thedevelopment of reproductive tissues. Binding of 17β-estradiol (E2) to ER induces a conformationalchange leading to dissociation of the receptor from the heat shock protein complex (hsp56-59,
hsp70 and hsp90). The activated ERα can interact with basal transcription factors (TFIIB, hTAF 30)
which permit RNA polymerase to initiate transcription of several genes such as: progesterone
receptor, ovalbumin, uteroglobin, lactoferrin, glucose-6-phosphate dehydrogenase and Xenopus
vitellogenin A2. Also, ERs are detected in 50-80% of all breast tumors and are clinically importantas markers for patients with a higher probability of responding to hormonal or endocrine treatments3. Therefore, accurate monitoring of ER activity in cells, tissues or animals is crucial forbiomedical research and drug development.
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 ER Kits are designed specifically for the study of ER expression and activity. They contain a 96-well plate to which an oligonucleotide containing the ER consensus binding site has been immobilized. ER contained in nuclear extracts binds specifically tothis oligonucleotide and is detected through use of an antibody directed against ERα. Addition of
a secondary antibody conjugated to horseradish peroxidase (HRP) provides sensitive colorimetricreadout that is easily quantified by spectrophotometry. The 96-well plate with individual strips of
8 wells is suitable for manual use or for high-throughput screening applications. TransAM ER Kits
are available in two sizes:
product
TransAM ER

formatcatalog no.

1 x 96-well plate5 x 96 well plates

41396
41996

See Active Motif products related to the ER transcription factor in Appendix, Section C.

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

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1Flow Chart of Process

4

3

2

1

5

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.
4

3

2

1

9

8

7

6

5

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.
2

1

5

4

3

7

6

A
B
C
D
E
F
G
H

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2

8

9

10

11

12

Introduction
ER Transcription Factor
ER belongs to the superfamily of ligand-inducible transcription factors. Human ERα is comprisedof 595 amino acids and displays an approximate molecular weight of 66-70 kDa. Six functional
regions have been identified4. A hypervariable domain (aa 1-184) contains activation function 1(AF1). The DNA binding domain (DBD, aa 185-263) contains two zinc finger motifs and is highly
conserved across the nuclear receptor superfamily5. It is responsible for the binding of thereceptor to estrogen response elements (EREs) and contributes to dimerization and activation5.
Typically, EREs consist of two inverted half-sites separated by 3 bp (5´-GGTCAnnnTGACC-3´)6. The
region which separates the ligand binding domain (LBD) and the DBD is called the hinge region(aa 264-302). The LBD (aa 303-553) consists of 12 α-helices, which form a hydrophobic pocket
responsible for ligand binding. The function of the final domain (aa 554-595) is not clear but isthought to play a role in distinguishing between agonist and antagonist binding7. Human ERβ is
expressed as multiple isoforms8. Structure and function studies have shown that the DBD of ERα
and ERβ are highly homologous, approaching 96%, whereas the LBD showed only 59% homology. The general mechanism of action of ERβ is thought to be similar to that of ERα. ERα and ERβ
have the ability to interact with target promoters in three different complexes: ERα homodimers,
ERβ homodimers and ERα/ERβ heterodimers.
The transcriptional effects of ER can be mediated through several mechanisms other than E2-ER
complexes binding to EREs. E2-ER complexes can also transactivate genes through proteinprotein interactions with transcription factors such as AP-1 or Sp1 that bind DNA, with coaccessory
proteins (Src, ACTR), some of which have histone acetylase activity, and with RNA Polymerase II
complex proteins9. In addition, ERs serve to repress genes, which also plays an important role inE2 action10, 11.

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

ER expression can be determined by Western blot by using antibodies specific for ER protein. 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 ER can be assayed by gel retardation, also called electrophoretic mobility shift assay (EMSA). In this method, nuclear extracts are incubated with aradioactive double-stranded oligonucleotide probe containing the consensus sequence for
ER binding. If ER is active in the nuclear extract, it will bind to the probe. Samples are thenresolved by electrophoresis on a native polyacrylamide gel, followed by autoradiography.
This method is sensitive, but like the previous procedure, it is time consuming (multiple daysof gel exposure may be required to achieve sufficient sensitivity) and it cannot be applied tohigh-throughput screening. Gelshift assays also require special precautions and equipment
for handling radioactivity.

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3

3.

Another method used to assay ER activation is based on reporter genes, typically luciferase
or β-galactosidase, placed under the control of a promoter containing an ER consensus
binding site. The promoter can be artificial, made of a GC box and a TATA box, or natural,
like promoter sequences from viral regulator elements, such as the HIV-1 LTR promoter.
However, the procedure is limited by the following issues: (i) reporter gene assays have tobe 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. 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 ER
ER-regulated genes are involved in a variety of cellular pathways that are currently being
deciphered by academic and pharmaceutical laboratories for new target discovery. However,this field has been hampered by the lack of convenient assays suitable for discriminating the ER
family members and performing high numbers of experiments.
To overcome this, Active Motif is introducing a high-throughput assay to quantify ER activ-ation12.
TransAM Kits combine a fast and user-friendly ELISA format with a sensitive and specific assay
for transcription factors. TransAM ER Kits contain a 96-well plate on which has been immobilized an oligonucleotide that contains an ER consensus binding site (5´-GGTCACAGTGACC-3´).
ER contained in nuclear extract specifically binds to this oligonucleotide. The primary antibody
used in the TransAM ER Kit recognizes an accessible epitope on ERα 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
ERα activation and has been shown to be 5-fold more sensitive and 20-fold faster than the gelretardation technique. With the 3.5-hour TransAM procedure, we could detect ER activation using
as little as 0.6 µg of nuclear extract from MCF-7 cells. A comparable assay using EMSA required
2.5 µg of nuclear extract and a 3-day autoradiography.
TransAM ER has many applications including the study of ER transcriptional activity regulationand protein structure/function studies of ER and its mutated variants in areas such as osteoporosis, arteriosclerosis, breast cancer propagation and many more.

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4Kit Performance and Benefits
Detection limit: < 0.6 µg nuclear extract/well. TransAM ER is 5-fold more sensitive than EMSA.
Range of detection: TransAM provides quantitative results from 0.6 to 20 µg of nuclear extract/well (see graph below).
Cross-reactivity: TransAM ER detects ERα from human, mouse and rat origin.

ER activation (OD450 nm)

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

3
2.5
2

H2O2 post-treated
Untreated

1.5
1
0.5
0

0.625

5
10
1.25
2.5
Nuclear extract (µg/well)

20

Monitoring ERα activation with the TransAM ER Kit: Different amounts of untreated and H2O2 post-treatednuclear extracts from MCF-7 cells are tested for ER activity by using the TransAM ER Kit. This data is 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 firstuse. Then, we recommend storing each component at the temperature indicated in the tablebelow. All components are guaranteed stable for 6 months from date of receipt when stored
properly.
Quantity
1 plate / 5 plates

StorageERα antibody

11 µl / 55 µl-20°C for 6 months

Anti-rabbit HRP-conjugated IgG

11 µl / 55 µl(0.2 µg/µl)

4°C for 6 months

Wild-type oligonucleotide AM11100 µl / 500 µl(10 pmol/µl)-20°C for 6 months

Mutated oligonucleotide AM11100 µl / 500 µl(10 pmol/µl)-20°C for 6 months

MCF-7 nuclear extract

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

Dithiothreitol (DTT)

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

Protease Inhibitor Cocktail

100 µl / 500 µl-20°C for 6 months

Poly [d(I-C)]

100 µl / 500 µl(17 µg/ml)-20°C for 6 months

Lysis Buffer AM1

10 ml / 50 ml

4°C for 6 months

Binding Buffer AM1

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 ER assay plate

1/5

4°C for 6 months

Plate sealer

1/5

Reagents

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 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 AM1 from the TransAM Kit. Prepare the amount of Complete Lysis Buffer required for theassay 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 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 required for the assay by adding 2 µl of DTT and
10 µl of 17 µg/ml poly[d(l-C)] per ml of Binding Buffer AM1 (see the Quick Chart for Preparing Buffers in this section). After use, discard remaining Complete Binding Buffer.
Preparation of 1X Wash Buffer
Prepare the amount of 1X Wash Buffer required for the assay as follows: For every 100 ml of 1X
Wash Buffer required, dilute 10 ml 10X Wash Buffer AM2 with 90 ml distilled water (see the Quick
Chart for Preparing Buffers in this section). Mix gently to avoid foaming. The 1X Wash Buffer may
be stored at 4°C for one week. The Tween 20 contained in the 10X Wash Buffer AM2 may formclumps, therefore it is necessary to completely resuspend any precipitates by incubating at 50ºC
for 2 minutes and mixing prior to use.
Preparation of 1X Antibody Binding Buffer
Prepare the amount of 1X Antibody Binding Buffer required for the assay as follows: For every
10 ml of 1X Antibody Binding Buffer required, dilute 1 ml 10X Antibody Binding Buffer AM3 with 9
ml distilled water (see the Quick Chart for Preparing Buffers in this section)*. Mix gently to avoid
foaming. Discard remaining 1X Antibody Binding Buffer after use. The BSA contained in the 10X
Antibody Binding Buffer AM3 may form clumps, therefore 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 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.
Nuclear extract
The MCF-7 nuclear extract is provided as a positive control for ER activation. Sufficient extract is
supplied for 20 reactions per plate. This extract is optimized to give a strong signal when used
at 5 µg/well. We recommend aliquoting the extract in 10 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 ER binding in order tomonitor the specificity of the assay. This competition assay will confirm that the protein subunits
binding to the plate are specific for the ER consensus binding sequence. Used at 20 pmol/well,
the oligonucleotide will prevent ER binding to the probe immobilized on the plate. Conversely,
the mutated consensus oligonucleotide should have no effect on ER binding. Prepare therequired 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 first, then add the cell extract. It is not necessary to perform an incubation
step of the oligonucleotide in the well prior to addition of the cell extract. The oligonucleotide
competition only needs to be performed as a control. It is suggested to test the oligonucleotide
competition each new cell type used.

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8Quick Chart for Preparing Buffers
For 1
well

For 1 strip(8 wells)

For 6 strips(48 wells)

For 12 strips(96 wells)

DTT
Protease inhibitor cocktail
Lysis Buffer AM1
TOTAL REQUIRED

0.01 µl0.12 µl11.12 µl11.25 µl

0.1 µl0.9 µl
89 µl
90 µl

0.6 µl5.4 µl534 µl540 µl1.2 µl10.8 µl
1.068 ml
1.08 ml

Complete Binding Buffer

DTT
Poly [d(l-C)]
Binding Buffer AM1
TOTAL REQUIRED

0.09 µl0.45 µl44.5 µl
45 µl

0.7 µl
3.6 µl356 µl360 µl

4.3 µl21.6 µl2.134 ml
2.16 ml

8.6 µl43.2 µl
4.268 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 µl

96 µl
2.064 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 µl2.25 ml

16.2 ml
1.8 ml18 ml

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

Distilled water
10X Ab Binding Buffer AM3
TOTAL REQUIRED

202.5 µl22.5 µl
225 µl1.62 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 µl

900 µl5.4 ml10.8 ml

Stop Solution

TOTAL REQUIRED

112.5 µl

900 µl5.4 ml10.8 ml

Reagents to prepare

Components

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

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9

ER Transcription Factor Assay
Determine the appropriate number of microwell strips required for testing samples, controls andblanks in duplicate. If less than 8 wells in a strip need to be used, cover the unused wells witha portion of the plate sealer while you perform the assay. The content of these wells is stable atroom temperature if kept dry and, therefore, can be used later for a separate assay. Store theunused strips in the aluminum pouch at 4°C. Use the strip holder for the assay.
Prepare the Complete Lysis Buffer, Complete Binding Buffer, 1X Wash Buffer and 1X Antibody
Binding Buffer as described above in the section Buffer Preparation and Recommendations.
Multi-channel pipettor reservoirs may be used for dispensing the Complete Binding Buffer, Wash
Buffer, Antibody Binding Buffer, Developing Solution and Stop Solution into the wells being used.

Step 1: Binding of ER 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. We recommend using 2-10 µg of nuclear extract diluted in Complete Lysis Buffer per well. A protocol
for preparing nuclear extracts can be found on page 13.
Positive control wells: Add 5 µg of the provided MCF-7 nuclear extract diluted in 10 µl ofComplete Lysis Buffer per well (2 µl of nuclear extract in 8 µl of Complete Lysis Buffer per well).
Blank wells: Add 10 µl Complete Lysis Buffer only per well.

Reagents

Blank
wells

Positive Control
no competition

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

40 µl
–
–
10 µl
–

40 µl
–
–
–
10 µl

40 µl
–
–
–
10 µl

38 µl
2 µl
–
–
10 µl

38 µl
–
2 µl
–
10 µl

3.

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

4.

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

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10Step 2: Binding of Primary Antibody1.

Add 100 µl diluted ERα antibody (1:2000 dilution in 1X Antibody Binding Buffer) to each wellbeing 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).

1.

Add 100 µl of diluted anti-rabbit HRP-conjugated antibody (1:2000 dilution in 1X Antibody
Binding Buffer) to all wells being used.

2.

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

3.

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

4.

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

1.

Add 100 µl Developing Solution to all wells being used.

2.

Incubate 5-10 minutes at room temperature protected from direct light. Please read theCertificate of Analysis supplied with this kit for optimal development time for this specific
kit lot. Monitor the blue color development in the sample wells until it turns medium to dark
blue. Do not overdevelop.

3.

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

4.

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

Step 3: Binding of Secondary Antibody

Step 4: Colorimetric Reactionwww.activemotif.com

11

References
1.

Cowley S.M., Hoare S., Mosselman S. and Parker M.G. (1997) Estrogen Receptors α and βForm Heterodimers on DNA. J. Biol. Chem. 272: 19858-19862.
2. Boris J., Cheskis J., Karathanasis S. and Lyttle C.R. (1997) Estrogen Receptor Ligands Modulate Its Interaction with DNA. J. Biol. Chem. 272: 11384-11391.
3. Traish A.M., Newton A.W., Styperek K., Beazley R. and Kavanah, M. (1995) Estrogen receptor
functional status in human breast cancer. Diag. Mol. Pathol. 4(3): 220-228.
4. Kumar V., Green S., Stack G., Berry M, Jin J.R., Chambon P., (1987) Functional domains of thehuman estrogen receptor. Cell 51: 941-951.
5. Greene G.L., Gilna P., Waterfield M., Baker A., Hort Y. and Shine J. (1986) Sequence andexpression of human estrogen receptor complementary DNA. Science 231:1150-1154.
6. Fawell S. E., Lees J. A., White R. and Parker M.G. (1990). Characterization and Colocalizationof Steroid Binding and Dimerization Activities In the Mouse Estrogen Receptor. Cell 60: 953962,
7. Brzozowski A.M., Pike A.C., Dauter Z. et al. (1997) Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389: 753-758.
8. Mosselman S., Polman J., Dijkema R. (1996) ER beta: identification and characterization of anovel human estrogen receptor. FEBS Lett. 392: 49-53.
9. McKenna N.J. and O’Malley B.W. (2000) From ligand to response: generating diversity innuclear receptor coregulator function. J Steroid Biochem. Mol. Biol. 74: 351-356.
10. Valentine J.E., Kalkoven E., White R., Hoare S. and Parker M.G. (2000) Mutations in the
estrogen receptor ligand binding domain discriminate between hormone-dependent transactivation and transrepression. J. Biol. Chem. 275: 25322-25329.
11. Wang Y, Miksicek R.J. 1991 Identification of a dominant negative form of the human estrogen receptor. Mol Endocrinol. 5:1707-1715.
12. Renard P., Ernest I., Houbion A., Art M., Le Calvez H., Raes M. and Remacle J. (2001) Nucleic
Acids Res. 29: (4) e21.

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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 toremove cells as it may alter transcription factor activation states.

3.

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

4.

Allow the cells to swell on ice for 15 minutes.

5.

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

6.

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

7.

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

8.

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

9.

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

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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 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 40 µg/well

Too many freeze/thaw
cycles of extract

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

ER is poorly activated or inactivated innuclear fractions

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

Nuclear extracts
are not from correct
species

Perform study with a human, mouse or rat 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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15

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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16