TransAM™ GR

Transcription Factor Assay Kits
(version A2)
Catalog Nos. 45496 & 45996

Information in this manual is subject to change without notice and does not constitute a commitment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any warrantyof any kind, either explicit or implied. Information may be changed or updated in this manual at
any time.
This documentation may not be copied, transferred, reproduced, disclosed, or duplicated, inwhole or in part, without the prior written consent of Active Motif, Inc. This documentation is proprietary information and protected by the copyright laws of the United States and international
treaties.
The manufacturer of this documentation is Active Motif, Inc.
© 2007 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
GR Transcription Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Transcription Factor Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
TransAM GR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Kit Performance and Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Protocols
Buffer Preparation and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
Quick Chart for Preparing Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
GR Transcription Factor Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Preparation of Nuclear Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Appendix Section A.
Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

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Overview
Glucocorticoids play an essential role in maintaining basal and stress-related homeostasis. Glucocorticoid Receptor (GR) is the mediator for most known effects of glucocorticoids.
Abnormalities in GR sensitivity can lead to steroid-resistant asthma, rheumatoid arthritis, osteoarthritis, hypertension and obesity1. Because of the importance of GR in maintaining homeostasis, accurate monitoring of GR in cells, tissues and animals is crucial for many biomedical
research and drug development projects. To date, such research projects are tedious and 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
with a sensitive, specific assay. TransAM GR Kits are designed specifically to detect and quantify
GR activation. They contain a 96-well plate to which oligonucleotide containing a GRE consensus sequence has been immobilized. GR contained in nuclear extract binds specifically to thisoligonucleotide and is detected through use of an antibody directed against GR. Addition of asecondary 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 GR Kits
are available in two sizes:
product
TransAM GR

formatcatalog no.

1 x 96-well plate5 x 96 well plates

45496
45996

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

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1Flow Chart of Process

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2

Introduction
GR Transcription Factor
Glucocorticoids can affect a large number of metabolic, cardiovascular, immune, inflammatory
and behavioral functions. They are produced by the adrenal cortex and are under the controlof the hypothalamus and pituitary (hypothalamus-pituitary-adrenal [HPA] axis). At the cellularlevel, glucocorticoid effects are mediated by the Glucocorticoid Receptor (GR). GR belongs to
the superfamily of nuclear hormone receptors that includes receptors for estrogens, progestins,
vitamin D and thyroid hormone1.
The nuclear hormone receptors share a characteristic three-domain structure. The N-terminal
activates target genes and interacts with transcription machinery. Two highly conserved zincfingers constitute the DNA-binding domain and also participate in dimerization, nuclear translocation and transactivation. The C-terminal contains the ligand-binding domain, and also includes
sequences important for heat shock protein (hsp) binding, nuclear translocation, dimerizationand transactivation1.
The unliganded GR is part of a multiprotein complex that consists of the receptor, two moleculesof hsp90 and one molecule each of hsp70 and hsp56. Glucocorticoids, when present, are able
to cross the cell membrane and interact with GR. When bound, there is a conformational change
in the GR molecule that results in dissociation from the hsp complex, hyper-phosphorylation of
GR and unmasking of nuclear localization signals. When in the nucleus, the activated GR can act
in two ways: directly with specific DNA sequences or indirectly with other transcription factors1.
GR mutations can result in glucocorticoid resistance or hypersensitivity, and can cause severe
disturbances in mood, pathologic alterations of metabolism and, correspondingly, hypotension
or hypertension and excessive or suppressed inflammatory/immune responses2.

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

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

2.

The DNA-binding capacity of GR 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 forGR binding. If GR 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
days of gel exposure may be required to achieve sufficient sensitivity) and cannot beapplied to high-throughput screening. Gelshift assays also require special precautions andequipment for handling radioactivity.

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3.

Another method used to assay GR activation is based on reporter genes, typicallyluciferase or β-galactosidase, placed under the control of a promoter containing the GR
recognition site. The promoter can be artificial, made of several GR elements or natural.
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, and 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 celltransfection with the reporter plasmid.

TransAM GR
GR plays a key role in the regulation of metabolism, cardiovascular function, inflammation andimmunity2, and therefore represents an excellent pharmacological target. However, pharmaceutical research in this field has been limited by the lack of convenient assays suitable for large
numbers of samples.
To overcome this, Active Motif is introducing a high-throughput assay to quantify GR activation.
The TransAM Kit combines a fast and user-friendly ELISA format with a sensitive and specificassay for transcription factors. TransAM GR Kits contain a 96-well plate on which has been immobilized an oligonucleotide that contains the GR consensus binding site (5´-GGTACAnnnTGTTCT-3´)3.
GR contained in nuclear extract binds specifically to this oligonucleotide. The primary antibody
used in TransAM GR Kits recognizes an accessible epitope on GR 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
GR activation and has been shown to be 5-fold more sensitive and 20-fold faster than the gel-retardation technique. With the 3.5-hour TransAM procedure, we could detect GR activation using
as little as 0.6 µg of nuclear extract. A comparable assay using EMSA requires 3 µg of nuclear
extract and a 5-day autoradiography.
TransAM has many applications including the study of drug potency toward GR, GR transcriptional activity regulation and protein structure/function studies of GR.

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4Kit Performance and Benefits
TransAM GR Kits are for research use only. Not for use in diagnostic procedures.
Detection limit: < 0.6 µg nuclear extract/well. TransAM GR is 5-fold more sensitive than EMSA.
Range of detection: TransAM provides quantitative results from 0.6 to 10 µg of nuclear extract/well (see graph below).
Cross-reactivity: The TransAM GR Kit detects GR from human, mouse and rat origin.

GR activation (OD450 nm)

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

1.6

Untreated
Dexamethasone treated

1.4
1.2
1
0.8
0.6
0.4
0.2
0

0.625

1.25
2.5
5
Nuclear extract (µg/well)

10

Monitoring GR activity with the TransAM GR Kit: Different amounts of nuclear
extracts from untreated and Dexamethasone treated HeLa cells are tested for GR
activity by using the TransAM GR 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.
Reagents

Quantity
1 plate / 5 plates

Storage / Stability

GR antibody

11 µl / 55 µl

4°C for 6 months

Anti-rabbit HRP-conjugated antibody

11 µl / 55 µl(0.25 µg/µl)

4°C for 6 months

Wild-type oligonucleotide AM27

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

Mutated oligonucleotide AM27

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

HeLa nuclear extract (Dexamethasone treated)

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

Herring sperm DNA

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

Lysis Buffer AM2

10 ml / 50 ml

4°C for 6 months

Binding Buffer AM2

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 GR assay plate

1/5

4°C for 6 months

Plate sealer

1/5

Additional materials required

For Nuclear Extract preparation• Multi-channel pipettor• Hypotonic Buffer• Multi-channel pipettor reservoirs• Phosphatase Inhibitor Buffer• Rocking platform• 10X PBS

• Microplate spectrophotometer capable of reading at 450 nm (655 nm as
optional reference wavelength)• Detergent (NP-40)

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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 cell 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 AM2 (see the Quick Chartfor Preparing Buffers in this section). Some of the protease inhibitors lose their activity after 24hours once diluted. Therefore, we recommend using the Complete Lysis Buffer immediately forcell 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 1 µl of 1 M DTT,
and 10 µl of 1 µg/µl Herring sperm DNA per ml of Binding Buffer AM2 (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, 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 atroom temperature for at least 1 hour. Transfer the amount of Developing Solution required forthe assay into a secondary container before aliquoting into the wells (see the Quick Chart for
Preparing Buffers in this section). After use, discard remaining Developing Solution.
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).

HeLa nuclear extract
The HeLa nuclear extract (Dexamethasone treated) is provided as a positive control for GR
activation. Nuclear extract was made from HeLa cells treated with 100 nM Dexamethasone for 1
hour at 37°C. Sufficient extract is supplied for 20 reactions per plate. 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
are available 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 GR binding in order tomonitor the specificity of the assay. Used at 40 pmol/well, the oligonucleotide will prevent GR
binding to the probe immobilized on the plate. Conversely, the mutated consensus oligonucleotide should have no effect on GR binding. Prepare the required amount of wild-type and/or
mutated consensus oligonucleotide by adding 2 µl of appropriate oligonucleotide to 31.8 µl ofComplete Binding Buffer per well being used (see the Quick Chart for Preparing Buffers in thissection). To allow for optimum competition, add the oligonucleotide to the well prior to additionof the cell extract.

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8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 AM2
TOTAL REQUIRED

0.02 µl0.23 µl22.25 µl22.5 µl

0.2 µl
1.8 µl
178.0 µl180.0 µl

1.2 µl10.8 µl
1.068 ml
1.08 ml

2.4 µl21.6 µl2.136 ml
2.16 ml

Complete Binding Buffer

DTT
Herring sperm DNA
Binding Buffer AM2
TOTAL REQUIRED

0.03 µl0.34 µl33.4 µl33.8 µl0.27 µl
2.7 µl267 µl270 µl

1.6 µl16.2 µl
1.6 ml1.62 ml3.25 µl
32.4 µl
3.2 ml3.24 ml

Complete Binding Buffer
with wild-type ormutated oligonucleotide

Wild-type or mutated oligo
Complete Binding Buffer
TOTAL REQUIRED

2.0 µl31.8 µl33.8 µl

16.0 µl254.0 µl270.0 µl96.0 µl1.524 ml
1.62 ml

N/A
N/A
N/A

1X Wash Buffer

Distilled water
10X Wash Buffer AM2
TOTAL REQUIRED

2.025 ml
225.0 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.

GR 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 stableat room 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.

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Step 1: Binding of GR to its consensus sequence1.

Add 30 µl Complete Binding Buffer to each well to be used. If you wish to perform competitive binding experiments, add 30 µl Complete Binding Buffer that contains 40 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 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 can be found on page 11.
Positive control wells: Add 5 µg of the provided 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 only 20 µl Complete Lysis Buffer per well.

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 GR 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).

Step 4: Colorimetric reaction1.

Add 100 µl Developing Solution to all wells being used.

2.

Incubate 2-20 minutes at room temperature protected from direct light. Please read theCertificate 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
and positive 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.

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10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 isapproximately 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
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.

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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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.

Bamberger C.M. et al (1996) Endocrine Reviews 17(3): 245-261.
Karl M. et al (1993) J. Clinical Endo and Metab. 76(3): 683-689.
Scott D.K. et al (1998) Mol. Endo. 12: 482-491.

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Appendix
Section A. Troubleshooting Guide
PROBLEMPOSSIBLE CAUSE

RECOMMENDATION

No signal or weak signal
in all wells

Omission of key reagent

Check that all reagents have been addedin the correct order

Substrate or conjugate is nolonger active

Test conjugate and substrate for activity

Enzyme inhibitor present

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

Plate reader settings not optimal

Verify the wavelength and filter settings in
the plate reader

Incorrect assay temperature

Bring substrate to room temperature

Inadequate volume ofDeveloping Solution

Check to make sure that correct volume
is delivered by pipette

Developing time too long

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

Concentration of antibodies
too high

Increase antibody dilutions

Inadequate washing

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

Incomplete washing of wells

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

Well cross-contamination

Follow washing recommendations

Too much nuclear extract per well

Decrease amount of nuclear extract down to5 µg/well

Concentration of antibodies
too high

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

Not enough nuclear extract per well

Increase amount of nuclear extract not toexceed 40 µg/well

GR is poorly activated orinactivated in nuclear fractions

Perform a time course for GR activationin the studied cell line

Extracts are not from rat,mouse or human origin

Perform study with a human, mouse or rat
modelHigh background in all wells

Uneven color development

High background in sample wells

No signal or weak signal insample wells

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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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